Compounds and methods for the detection of trpv-6 cancers and drug delivery

ABSTRACT

Compounds containing TRPV6-binding peptides and their use in the detection and diagnosis of cancer are described. Also described are methods for detecting and staging cancer that use the compounds of the invention. Compounds containing TRPV6-binding peptides are useful for the delivery of diagnostic and therapeutic agents to cells or tumors that express TRPV6.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/046,786, filed Jul. 26, 2018; which is a continuation of U.S.application Ser. No. 14/579,157, filed Dec. 22, 2014, now U.S. Pat. No.10,064,964, issued Sep. 4, 2018; which is a continuation of U.S.application Ser. No. 12/824,935, filed Jun. 28, 2010 (abandoned); whichclaims priority to U.S. application No. 61/220,833 filed on Jun. 26,2009 and to U.S. application No. 61/244,634 filed on Sep. 22, 2009, allof which are incorporated by reference herein in their entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing “SORI_001_05US_ST25.txt” (2 KB), submitted via EFS-WEB and created on Jul. 14, 2021,is herein incorporated by reference.

FIELD

The present invention relates to detection and diagnosis of TransientReceptor Potential Vanilloid 6 (TRPV6)-expressing cancers. Certainembodiments of the invention also relate to TRPV-6 binding compoundsthat target cells that express TRPV6 for use in diagnostics or drugdelivery.

BACKGROUND

Soricidin (NCBI accession no. P0C2C6) is a fifty-four amino acidparalytic peptide isolated from the submaxilary saliva gland of theNorthern Short-tailed Shrew (Blarina brevicauda). U.S. Pat. No.7,119,168 (incorporated by reference herein in its entirety) describessoricidin, its paralytic activity and usefulness of the peptide forconditions, such as treating pain and neuromuscular disease. U.S. Pat.No. 7,273,850 (incorporated by reference herein in its entirety)describes that soricidin has paralytic activity, and among other things,provides data that it inhibits calcium uptake in two ovarian cancer celllines.

One group of calcium ion channels implicated in cancer is the TransientReceptor Potential (TRP) channels that are found across theinvertebrates and vertebrates. The Transient Receptor PotentialVanilloid (TRPV) members of the TRP super-family were named after it wasdiscovered that they activate in the presence of vanilloids (capsaicinfrom hot peppers for example). The first four of these receptors tested(TRPV1, TRPV2, TRPV3 and TRPV4) all responded to capsaicin and were alsoresponsible for detecting changes in temperature and other environmentalsignals. The remaining two of the TRPV sub-family, TRPV5 and TRPV6, werefound predominantly in epithelial type or derived tissues and wereresponsible for influx of calcium ion into the cell. U.S. Pat. No.7,7205,108 describes genes encoding TRPV 8, 9 and 10 and their use asbiomarkers for cancer and in associated diagnostic and therapeuticmethods.

TRPV6 was identified as responsible for import of calcium intoepithelial tissues of the intestine and hence uptake of calcium from thediet. These channels were also shown to be present in a number of othertissues in varying amounts, but most notably intestinal epithelial cell,kidney, placenta and pancreas. The expression of TRPV6 was measured ashighly elevated in some cancer tissues and in some known ovarian,breast, prostate, and leukemia cancer cell-lines. (Peng et al. 2000;Zhuang et al. 2002).

Accordingly, there is a need for compounds and associated methods fordetecting cells that express TRPV6. There is also a need forcompositions and methods capable of targeting cells that express TRPV6for the delivery of agents useful for the diagnosis or treatment ofcancer.

SUMMARY

The present inventor has synthesized compounds that bind to TRPV6calcium ion channels. These compounds have at least a portion that hassequence identity to a continuous string of amino acids from theC-terminal peptides of soricidin. In certain cases, the peptidecomponent represents the entirety of the compound, while in other casesthe peptide is a component of the compound, for example, if the compoundcomprises peptide conjugated to a drug or a detectable label.

It was not previously known that the structure of soricidin thatprovided calcium channel inhibition activity was separate from thestructure that caused paralytic activity. Fluorescent conjugates of thecompounds described herein bind to cells expressing TRPV6 protein inco-localization experiments with TRPV6 antibodies. TRPV6 has been shownto be overexpressed in a number of cancer-tissue samples and cell lines.TRPV6-binding compounds are useful for the identification of cancer aswell as for targeting anti-cancer drug activity to cells that expressTRPV. A further aspect includes the use of TRPV6 antibodies in theidentification and diagnosis of cancer as well as for targetinganti-cancer drug activity to cells that express TRPV. In addition,fluorescently labeled TRPV6-binding compounds have been shown to beuseful for imaging and identifying tumors in vivo. The TRPV6-bindingpeptides described herein have also been shown to be useful fortargeting biomolecules to cells expressing TRPV6, such as tumor cells.

Accordingly, some embodiments include a compound comprising a TransientReceptor Potential Vanilloid 6 (TRPV6)-binding peptide conjugated to abiomolecule. Optionally, the compound may comprise all or part of apeptide with the amino acid sequence EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ IDNO:1). In one embodiment, the compound comprises from 9 to 27 aminoacids of SEQ ID NO:1. In one embodiment, the compound comprises acontiguous part of the C-terminal sequence of SEQ ID NO:1. In oneembodiment, the compound comprises at least 9 contiguous amino acids ofSEQ ID NO:1, at least 10 contiguous amino acids of SEQ ID NO:1 orgreater than 10 contiguous amino acids of SEQ ID NO:1. In oneembodiment, the compound comprises a TRPV6-binding peptide with at least70%, at least 80%, or at least 90% identity to one of HPSKVDLPR,KEFLHPSKVDLPR or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO:1). In someembodiments, the TRPV6-binding peptide comprises the amino acid sequenceHPSKVDLPR or KEFLHPSKVDLPR.

In some embodiments, there is provided a compound comprising an antibodyto TRPV6 conjugated to a biomolecule.

In one embodiment, the compound comprises a biomolecule with adetectable label. In some embodiments, the biomolecule is fluorescently,radioactively or immunologically labeled. In one embodiment, thedetectable label comprises a magnetic resonance imaging (MRI) contrastagent. In one embodiment, the detectable label is super-paramagneticiron oxide (SPIO).

In one embodiment, the biomolecule is a therapeutic agent. Optionally,the therapeutic agent is an anti-cancer agent for example a taxane-baseddrug, anthracycline-type drug or a platin-based drug.

In some embodiments, the biomolecule is a small drug molecule,oligosaccharide, antibody, antibody epitope, nanometallic cluster,radioactively-labeled molecule, taxane-based drug, anthracycline-typedrug, platin-based drug, antibiotic, anti-cancer drug, anti-fungal,anti-viral or anti-retroviral, or boron complex, epitope for anendogenous or therapeutically administered antibody, signaling peptideor oligosaccharide that recruits immune cells e.g. killer-T cells.

In one embodiment, the TRPV6-binding peptide and the biomolecule areattached through a spacer. In one embodiment, the TRPV6-binding peptideis conjugated to more than one biomolecule or to more than one type ofbiomolecule.

Also included are pharmaceutical compositions comprising a compoundcontaining a TRPV6-binding peptide as described herein and apharmaceutically acceptable carrier. Optionally, the pharmaceuticalcomposition comprises a TRPV6-binding peptide conjugated to abiomolecule, or to a plurality of biomolecules.

One embodiment includes a method for detecting TRPV6 protein in a samplecomprising contacting the sample with a TRP-binding peptide comprisingall or part of a peptide comprising EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ IDNO:1) and detecting the TRPV6-binding peptide. In one embodiment, theTRPV6-binding peptide is detected using an antibody that selectivelybinds the TRPV6-binding peptide.

Another embodiment includes a method for detecting TRPV6 protein in asample comprising contacting the sample with a compound comprising aTRPV6-binding peptide conjugated to a biomolecule and then detecting thebiomolecule. For example, in one embodiment the biomolecule is afluorophore and the compound comprising the TRPV6-binding peptide isdetected by detecting the fluorophore.

A further embodiment includes the use of the compounds described hereinfor detecting TRPV6 in a sample. In one embodiment, the sample is abodily fluid such as blood, saliva or urine.

Some embodiments relate to methods for identifying cancer in a samplefrom a subject comprising detecting TRPV6 mRNA or protein in the sampleand comparing the amount of TRPV6 mRNA or protein in the sample with acontrol sample, wherein an increased amount of TRPV6 mRNA or protein inthe sample compared to the control is indicative of cancer. In oneembodiment, the sample is a bodily fluid. In some embodiments, the TRPV6protein is detected in vivo, ex vivo or in vitro. The TRPV6 protein canbe detected using the compounds described herein or antibodies directedto TRPV6. In some embodiments, TRPV6 mRNA is detected using PCR, RT-PCRor real time quantitative RT-PCR. In some embodiments, said cancer is anearly stage cancer such as stage I or stage II cancer.

In some embodiments the subject can be a mammal, such as a human. Thesample may comprise a bodily fluid, excreta, tissue sample, tumor sampleor microvesicles. In one embodiment, the bodily fluid is blood, urine,saliva, plasma, cerebrospinal fluid, mucus, vaginal secretions, lymph orpleural fluid.

In some embodiments the methods described herein are used to identifybreast cancer, ovarian cancer, blood cancer, brain cancer, retinalcancer, liver cancer, thyroid cancer, colon cancer, prostate cancer,pancreatic cancer, glial cancer, leukemia or endometrial cancer. In oneembodiment, the cancer is metastatic cancer or lymph node metastaticcancer.

In one embodiment, the methods described herein are used to stagecancer. In some embodiments, the method includes comparing the amount ofTRPV6 mRNA or protein in a sample with a control sample or samples thathave stage I, II, III or IV cancer.

In another embodiment, the methods described herein are used to gradecancer cells or tumors. In some embodiments, the cancer is ovariancancer. In one, the methods disclosed herein identify subjects withgrade I ovarian cancer.

A further embodiment includes a method of manufacturing one of thecompounds disclosed herein comprising conjugating a biomolecule to aTRPV6-binding peptide or to a TRPV6 antibody. In one embodiment, theTRPV6-binding peptide is covalently conjugated to a biomolecule. Forexample, in one embodiment the TRPV6-binding peptide comprises all orpart of SEQ ID NO:1 and the biomolecule is attached through the cysteinthiol corresponding to position 14 in SEQ ID NO:1. In one embodiment,the biomolecule is conjugated to the peptide through an activatedmaleimide.

Yet another embodiment includes a method of delivering a biomolecule toa cell expressing TRPV6 comprising contacting the cell with a compoundcomprising a TRPV6-binding peptide conjugated to a biomolecule or aTRPV6 antibody conjugated to a biomolecule. In one embodiment, thebiomolecule comprises a detectable label or a therapeutic agent. Thestep of contacting the cell with the compound can occur in vivo, invitro or ex vivo. In some embodiments the cell expressing TRPV6comprises a tissue, tumor or microvesicle.

Additional embodiments include kits for detecting TRPV6 in a samplecomprising reagents for conducting the methods described herein andinstructions for use. Other embodiments include kits for diagnosingcancer, reagents for conducting the methods described herein andinstructions for use.

In another aspect, there is provided a method of identifying a cancertumor in a subject. In one embodiment, the cancer tumor over-expressesTRPV6. In some embodiments, the method comprises administering to thesubject a compound comprising a TRPV6-binding peptide or an antibody toTRPV6 as described herein. The TRPV6-binding peptide or antibody toTRPV6 is then detected, indicating the presence of TRPV6. In oneembodiment, the TRPV6-binding peptide is detected by detecting abiomolecule conjugated to the peptide. Optionally, regions of thesubject with increased levels of TRPV6 are then identified, whereinincreased levels of TRPV6 are indicative of a tumor. In one embodiment,the levels TRPV6 are compared to a control level such as those observedin a non-cancerous tissue, a pre-determined control level, or an averagelevel taken throughout the subject. In one embodiment, TRPV6 is detectedin vivo, for example by detecting a fluorescent label conjugated to theTRPV6-binding peptide or antibody to TRPV6. In one embodiment, TRPV6 isdetected using magnetic resonance imaging (MRI) and an MRI contrastagent conjugated to a TRPV6-binding peptide. In some embodiments, thecancer tumor is a prostate tumor, a breast tumor or an ovarian tumor.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described in relation to thedrawings in which:

FIG. 1 is a line drawing showing the location of the lymph nodes in themouse. Significant accumulation of SorC13-Cy5.5 compound andSorC27-Cy5.5 compound four hours after i.v. injection of 100 ug of eachof the labeled peptides into CD1 mice was observed in the followingnodes labeled in FIG. 1: 1. Superfacial cervical nodes; 4. Axillarynodes; 5. Branchial nodes; 8. Mesenteric nodes; 9. Inguinal nodes.

FIG. 2 shows the distribution of SorC13-Cy5.5 compound in CD1 mice 4hours after i.v. injection. The Y-axis is the percentage of totalfluorescence measured in all tissues.

FIG. 3 shows the distribution of SorC27-Cy5.5 compound in CD1 mice 4hours after i.v. injection. The Y-axis is the total fluorescencemeasured in each tissue.

FIG. 4 shows the distribution of SorC13-Cy5.5 compound in CD1 mice afteri.v. injection over time after perfusion to wash out fluids. The Y-axisis the percentage of total fluorescence measured in all tissues. Thehighest percent uptake (of total fluorescence) of SorC13-Cy5.5 compoundwas observed in liver, lung and kidney. Lymph node is not shown becauseperfusion washes out lymph. 4A shows the distribution 4 hours after i.v.injection and 4B 24 hours after i.v. injection.

FIG. 5 shows the immuno-localization of TRPV6 in ovarian cancer cells(SKOV-3). SKOV-3 cells were transfected with a TRPV6-GFP fusion protein.Both endogenous TRPV6 and TRPV6-GFP were detected with a combination ofa primary antibody to TRPV6 N-terminal region and a secondary antibodyto IgG labeled with FITC (a green fluorescent label). One cell in thefield shows co-localization of brightly fluorescent, transfectedTRPV6-GFP protein and the antibody to TRPV6. The other cells (nottransfected) in the field show the red fluorescence of wheat germagglutinin marking the cell membrane, and the green fluorescence ofimmuno-localization indicating the secondary antibody labeled with FITCused to detect the primary anti-TRPV6 antibody.

FIG. 6 shows the co-localization of TRPV6 protein expressed in HEK-293cells with antibody to TRPV6 and with SorC27-cy5.5. HEK293 cells nottransfected with the TRPV6 expression vector show no fluorescence whenincubated with SorC27-cy5.5 (FIG. 6A, negative control). FIG. 6B showscells imaged with green TRPV-antibody while FIG. 6C shows the same fieldof cells imaged with red SorC27-cy5.5. FIG. 6D shows both imagessuperimposed and the co-localization of TRPV6 and SorC27-cy5.5 in cellstransfected with TRPV6 vector.

FIG. 7 shows the co-localization of SorC27-cy5.5 and fluorescentlylabeled TRPV6 antibodies in prostate cancer cell line PC-3. The A1-A3series of images shows A1: TRPV6-antibody immunofluorescence, A2:labeling with SorC27-cy5.5 and A3: overlap of A1 and A2. The B1-B3series of images shows PC-3 cells in the same sequence as the A1-A3series, but now transfected with a TRPV6 expression vector to increasethe level of TRPV6 expressed by the cells. Both series of images showco-localization of SorC27-cy5.5 and TRPV6. The level of TRPV6immunofluorescence and SorC27-cy5.5 fluorescence are both increased inthe transfected cells with increased TRPV6 expression shown in the B1-B3series of FIG. 7 compared to the cells in the A1-A3 series of FIG. 7.

FIG. 8 shows the co-localization of SorC27-cy5.5 and fluorescentlylabeled TRPV6 antibodies in breast cancer cell line T 47D. The A1-A3series of images shows A1: immunofluorescence, A2: labeling withSorC27-cy5.5 and A3: overlap of A1 and A2. The B1-B3 series of imagesshows PC-3 cells in the same sequence as the A1-A3 series, but nowtransfected with a TRPV6 expression vector to increase the level ofTRPV6 expressed by the cells. Both series of images show co-localizationof SorC27-cy5.5 and TRPV6. The level of TRPV6 immunofluorescence andSorC27-cy5.5 fluorescence are both increased in the transfected cellswith greater TRPV6 expression shown in the B1-B3 series of FIG. 8compared to the cells in the A1-A3 series of FIG. 8.

FIG. 9 shows the co-localization of SorC27-cy5.5 and fluorescentlylabeled TRPV6 antibodies in ovarian cancer cell line SKOV-3. The A1-A3series of images shows A1: immunofluorescence, A2: labeling withSorC27-cy5.5 and A3: overlap of A1 and A2. The B1-B3 series of imagesshows PC-3 cells in the same sequence as the A1-A3 series, but nowtransfected with a TRPV6 expression vector to increase the level ofTRPV6 expressed by the cells. Both series of images show co-localizationof SorC27-cy5.5 and TRPV6. The level of TRPV6 immunofluorescence andSorC27-cy5.5 fluorescence are both increased the transfected cells withgreater TRPV6 expression shown in the B1-B3 series of FIG. 9 compared tothe cells in A1-A3 series of FIG. 9.

FIG. 10 shows the PCR detection of TRPV6 cDNA present in cDNA librariesmade from total RNA extracted from a number of human ovarian tumorbiopsies. Lane 1: Blank; 2: LTL320 TRPV6; 3: LTL320 ß-actin; 4: LTL317TRPV6; 5: LTL31713 ß-actin, 6: LTL269 TRPV6; 7: LTL269 ß-actin, 8: 100base pair ladder; 9: TRPV6 negative control; 10: ß-actin negativecontrol, The ranking of the band densities of the 3 TRPV6 amplicons foras reported in Table 1 is: LTL320 (+); LTL317 (+++); LTL269 (++++). TheTRPV6 amplicon is at approximately 370 base pairs and the ß-actinamplicon is at 50 base pairs.

FIG. 11 shows the PCR detection of TRPV6 cDNA present in cDNA librariesmade from total RNA extracted from a number of human ovarian cancer celllines. Lane 1—Blank; 2—100 base pair ladder; 3—Positive control;4—OVCAR3; 5—SKOV3 6A—OVC13 badly degraded, 6B OVC13 repeat showing someTRPV6 signal; 7—HEYC2; 8—OV2008; 9—Negative control; 10—100 bp ladder.The TRPV6 amplicon is at approximately 370 base pairs. Lanes 7 and 8were isolated from very small sample sizes (low cell count) and thusshow weak signals.

FIG. 12A shows the PCR detection of TRPV6 cDNA present in cDNA librariesmade from total RNA extracted from a number of human breast cancer celllines. Lane 1: HCC1954 ß-actin; 2: T47D ß-actin; 3: MCF10A ß-actin; 4:100 base pair ladder; 5: HCC1954 TRPV6; 6: T 47D TRPV6; 7: contaminatedsample; 8: TRPV6 positive control from a TRPV6-containing expressionvector (pcAGGS-TRPV6). FIG. 12B shows the PCR detection of TRPV6 cDNApresent in cDNA libraries made from total RNA extracted from the humanbreast cancer cell line MB423 (to the right of the 100 bp ladder). TheTRPV6 amplicon is at approximately 370 base pairs.

FIG. 13 shows PCR detection of TRPV6 cDNA present in cDNA libraries madefrom total RNA extracted from a human prostate cancer cell line (PC-3).The TRPV6 amplicon is at approximately 370 base pairs. The molecularweight ladder is a 100 bp ladder. Lane 1: Blank; Lane 2: PCR of cDNAlibrary from PC-3 cells; Lane 3: blank; Lane 4: MW ladder; Lane 5:blank.

FIG. 14 is a Western blot showing the detection of TRPV6 proteinover-expression in extracts from human ovarian, breast and prostatecancer cell lines. Lane 1: Molecular weight standard; 2: blank; 3: HEPG2 (positive control); 4: Breast cancer cell line T 47D; 5: Ovariancancer cell line SKOV-3; 6: Prostate cancer cell line PC-3. In lane 3,the HEP G2 (hepatoblastoma) lysate shows two bands: the top band isTRPV6 that has not been glycosylated while the fully glycosylated TRPV6is shown in the second band. The de-glycosylated TRPV6 is heavilyproduced in all three cancer cell types. De-glycosylation ofmembrane-bound TRPV6 has been shown to trap the ion channel in themembrane and to increase channel activity (Lu et al., 2008).

FIGS. 15A, 15B, 15C and 15D are Western blots showing the detection ofTRPV6 protein in extracts from 18 human ovarian tumor samples. Eachseparate patient/tumour is cited as an alphanumeric code. In allsections the top arrow at the right of the image indicates the positionof the glycosylated form of TRPV6 while the lower arrow indicates theposition of the de-glycosylated form of TRPV6 protein. FIG. 15A: Lane 1:molecular weight standard; 2: LTL-175; 3: LTL-205; 4: LTL-234; 5:LTL-237; 6: LTL-246; 7: HEP G2 (positive control); 8: molecular weightstandard. FIG. 15B: Lane 1: molecular weight standard; 2: LTL-247; 3:LTL-258; 4: LTL-259; 5: LTL-260; 6: LTL-269; 7: HEP G2 (positivecontrol); 8: molecular weight standard. FIG. 15C: Lane 1: molecularweight standard; 2: LTL-273; 3: LTL-284; 4: LTL-290; 5: LTL-300; 6:PC-3; 7: blank; 8: molecular weight standard. FIG. 15D: Lane 1:molecular weight standard; 2: LTL-305; 3: LTL-315; 4: LTL-317; 5:LTL-320; 6: PC-3; 7: blank 8: molecular weight standard. The PC-3 was asmall protein load (50 ug) to match the ovarian tumor biopsy proteinload (50 ug) and shows a very weak signal, barely visible in 15D (forexample see FIG. 14, lane 3 for HEP G2 and lane 6 for PC-3). Trapped,de-glycosylated TRPV6 is the predominant form observed in each ovariantumor sample tested. The bands in the HEP-G2 control are very faint withthis amount of loaded protein and indicate further the over-expressionof TRPV6 in the tested biopsies.

FIG. 16 is a Western blot showing the detection of TRPV6 proteinover-expressed in extracts of human glioblastoma (U87MG), human colon(CaCo-2) and pancreatic carcinoma cells (Panc1). Lane 1: Molecularweight markers with the light thin band at 75 kDa; 2: U87MG cells; 3:CaCo-2 cells; 4: Panc1 cells at 2 culture passages; 5: Panc1 cells at 5culture passages; 6: Panc1 cells at 7 culture passages. In the lastthree lanes, increasing the passage number appears to increase theamount of de-glycosylated TRPV6. The top band is the glycosylated formof the ion channel and the lower band is the de-glycosylated form ofTRPV6.

FIG. 17 shows the calibration of grading for tissue micro-arrayimmunohistochemical (IHC) scores with corresponding representativesample images.

FIG. 18 shows the percentage of tissue micro-array slides that werenegative for TRPV6 antibody staining or had a stain intensity score 1for normal ovarian tissue samples compared to serous papillaryadenocarcinoma tissue samples with grade I, grade II, or grade IIIcancer. 100% of serous papillary adenocarcinoma tissue samples had astain intensity score of 1, compared with only about 24% of normalovarian tissues.

FIG. 19 shows the immunohistochemical detection of TRPV6 in micro-arraysamples of normal ovarian tissues, as well as in samples of Grades I, IIand III serous papillary carcinoma. TRPV6-antibody staining intensityscores (−, −/+, +, ++, +++ or ++++) are also given for each sample.

FIG. 20 shows the co-localization of antibodies to TRPV6 andfluorescently labeled SorC27-cy5.5 in a tissue micro-array sample ofgrade II serous papillary adenocarcinoma. 20A shows an ovarian tumourbiopsy stained with an antibody to TRPV6 while 20B shows the same samplestained with the fluorescent tagged peptide SOR-C27-cy5.5.

FIG. 21 shows the time dependent localization of SorC27-cy5.5 inxenograft mouse models of ovarian (FIG. 21A) and prostate (FIG. 21B)cancer tumors. FIG. 21C shows: 3-D image of a mouse indicating the planeof observation (left), an image of a 2 mm slice of the mice through thecenter (middle) and, an image showing a perpendicular ‘slice’ throughthe center of the tumors (right) for both ovarian (SKOV-3; top) andprostate (DU145; bottom) tumors.

FIGS. 22A-22C show TRPV6 mRNA expression relative to healthy controls insamples of ovarian (22A), prostate (22B) and breast (22C) cancer.

FIG. 23 shows the localization of an MRI enhancement agent conjugated tothe TRPV6 binding peptide SorC27 (SPIO-SorC27) to SKOV-3 derived ovariantumors xenografted into CD-1 nude mice. 23A shows the tumor indicated bythe white arrow prior to and 24 hours after injection of SPIO controlbeads. 23B shows the tumor prior to (white arrow) and 24 hours afterinjection with SPIO-SorC27 and the localization of the contrast agent tothe tumor site (dashed white arrow).

FIGS. 24A-24C show RT-PCR analysis of the amount of TRPV6 mRNA isolatedfrom blood samples from healthy controls and patients with prostate(FIG. 24A), breast (FIG. 24B) and ovarian (24C) cancers at differentcancer stages. RT-PCR of TRPV6 mRNA easily distinguishes subjects withstage I cancer from healthy controls.

FIG. 25 shows the amount TRPV6 mRNA determined by RT-PCR in blood plasmaof healthy women compared to women with either Stage I or Stage IIovarian cancer. The data represent the integrated band density readingsof the amplicons from the samples. The samples were determined intriplicate for each sample. Both Stage I (p<0.0001) and Stage II(p=0.047) TRPV6 levels were statistically significantly larger thanobserved in plasma from healthy women.

FIG. 26A shows Western blot data for TRPV6 protein levels in samples ofblood plasma from healthy women compared to women with Stage I and StageII ovarian cancer. The figure shows the quantified band density of TRPV6antibody staining. Both Stage I (p=0.0001) and Stage II (p=0.0210) TRPV6levels were significantly higher than those observed in plasma fromhealthy women. FIG. 26B compares the band density from Western Blots ofhealthy plasma and combined Stage I and II data (‘early stage’ ovariancancer). The plasma from combined stage I and II cancer patientscontained statistically significant higher amounts of TRPV6 protein thanhealthy women (p=0.0006).

DETAILED DESCRIPTION

The inventor has determined that TRPV6 calcium channel expression isupregulated in certain cells, such as ovarian cancer cells, and thatthis overexpression is indicative of cancer. The present descriptionprovides new methods for detecting TRPV6 overexpression for identifyingand/or diagnosing cancer. The application also provides new compoundsand methods that target TRPV6 protein or cells that express TRPV6 suchas cancer cells.

In one embodiment, the inventor synthesized compounds that bind tocalcium channels and in particular to TRPV6 calcium channels. TheTRPV6-binding compounds described herein have sequence identity to partof soricidin but do not exhibit paralytic activity. It is surprisingthat the compounds retain TRPV-6 binding activity in the absence ofparalytic activity. It was not previously known that soricidin has twofunctional domains in its structure, one portion that binds to calciumchannels and the other portion which binds to sodium channels. It wasalso unknown that peptides could be prepared that separated the calciumchannel detection/binding activity from the sodium-channel bindingparalytic activity.

The inventor has determined that it is an N-terminal domain of soricidinthat has the paralytic function and a C-terminal domain that has thecalcium channel inhibitor function, and more specifically TRPV6-bindingactivity. Truncating soricidin at the N-terminal successfully producedpeptides that retain calcium channel binding/detection activity withoutexhibiting paralytic activity. The compounds therefore are more likelyto bind to TRPV6 in vivo because sodium channels cannot bind thecompounds and remove them from circulation. As well, the calciumchannel-binding activity is now obtained without the unwanted sideeffect of sodium channel-binding, which avoids paralysis and otherpotential side effects. The surprising nature of the embodimentsdescribed herein is emphasized by considering that, while bifunctionallarge proteins and enzymes are common in biological systems, inherentbifunctionality is a very rare phenomenon in small peptides,particularly where one end of the peptide binds calcium channels and theother end of the peptide binds sodium channels. Reports in theliterature of bifunctionality have typically resulted from artificialproduction, for example, where two different peptides have beenchemically fused (Anes et al. 2006; Yamamoto et al. 2008).

The compounds described herein typically have a peptide component thatis half the length of soricidin, or shorter. In one embodiment, thepeptide retains TRPV6 binding activity. In some embodiments, the peptidecomponent is the entirety of the compound, while in other embodiments itis a component of the compound, for example, if the compound comprisespeptide conjugated to a drug or a detectable label. In one embodiment,the TRPV6-binding peptide Comprises all or part of a peptide comprisingEGKLSSNDTEGGLCKEFLHPSKVDLPR (called “SorC27”; SEQ ID NO:1). TheTRPV6-binding peptide optionally comprises a contiguous part of SEQ IDNO:1. In some embodiments, the TRPV6-binding peptide comprises acontiguous part of the C-terminal sequence of SEQ ID NO:1. Optionallythe TRPV6-binding peptide comprises at least: 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 amino acids of SEQ ID NO:1. In some embodiments,the compound comprises a TRPV6-binding peptide that has at least 50%identity over its full length to a part of SEQ ID NO:1. In someembodiments, the compound comprises a TRPV6-binding peptide that has atleast 70%, 80% or 90% identity over its full length to a part of SEQ IDNO:1. In some embodiments, the TRPV6-binding peptide optionallycomprises, consists essentially of or consists of the amino acidsequence: HPSKVDLPR (called “SorC9”; amino acid nos. 19-27 of SEQ IDNO:1), KEFLHPSKVDLPR (called “SorC13”; amino acid nos. 15-27 of SEQ IDNO:1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (“SorC27”; SEQ ID NO:1). The SorC99 amino acid sequence HPSKVDLPR contains three positive charges atphysiological pH that are expected to interact with the four negativelycharged aspartic acids that are present at the entrance to the calciumchannel of the TRPV6 tetramer (den Dekker et al. 2003).

In some embodiments, the TRPV6-binding peptides are typically 35 or 30amino acids or less, optionally less than: 27, 25, 20, 15 or 13 aminoacids long, while optionally at least 9 amino acids long. TheTRPV6-binding peptide optionally comprises at least 9-13, 10-15, 15-20,20-25 or 20-27 amino acids. In some embodiments, amino acids may beadded to the TRPV6-binding peptides described herein. One can readilymake longer peptides by adding a variety of additional amino acids tothe SorC27 sequence to make a TRPV6-binding peptide that could be up to,for example, 30, 35, 40 or 45 amino acids long (e.g. additional aminoacids corresponding to the soricidin amino acid sequence such as one ormore of the amino acids that are immediately towards the N-terminalsegment of SorC27 in soricidin (SILARPAELNTETCILEC SEQ ID NO:2), atargeting sequence, or other amino acids) or longer.

In some embodiments, the compounds comprise TRPV6-binding peptideconjugated to a biomolecule, which is optionally a second protein orpeptide, either directly or through a spacer. In some embodiments, thespacer molecule may be a carbon containing substance. For example, thespacer may be a short peptide, such as 3 amino acids (Gly-Gly-Gly) orlonger, or a carbon chain, for example, (CH₂)_(n) wherein n optionallyequals 1 to 5, 1 to 10, or greater than 10. In other embodiments thespacer is optionally a polymer, for example Polyethylene Glycol (PEG), asugar, a lipid or the like.

As noted above, a TRPV6-binding peptide of the invention will havecalcium channel binding activity. The peptides having such activity arereadily identified with any known assays suitable for measuring bindingof the peptides with TRPV6. For example, in one embodiment, a peptidehaving calcium channel inhibition activity is optionally identified bydetermining that the peptide reduces calcium channel activity byreducing (i.e., partially or fully inhibiting) the flow of calciumthrough calcium channels. TRPV6-binding activity can also be measuredusing methods such as: internalized calcifluors (such as FURA-2 am);whole cell patch clamping to measure inhibition calcium currentselectrophysiologically; equilibrium binding assays using peptideslabeled with detectable tags such as radioactive or fluorescent tags;and/or NMR determination of ‘bound’ and ‘unbound’ peptides. In someembodiments, the TRPV6-binding peptides also have i) calcium channelblocking and ii) lack of paralytic activity.

Calcium channel binding activity is optionally identified by using areadily available cell line (e.g. human embryonic kidney celllines-HEK293) transfected with an expression vector for TRPV6. Suchtransfected cells are readily aliquoted and stored (typically −80° C.)until required. This provides a standard transfected cell preparation totest for detection of calcium ion channels in the cells and for testingfor the inhibition of these ion channel activities. Optionally thepeptides have an equilibrium inhibition constant of less than: 1000 nM,150 nM or 100 nM in LNCaP, HEK293 or SKOV-3 cell models. For example,the equilibrium dissociation constant (Kd) for SorC27 is 140 nM and forSorC13 is 100 nM in an LNCaP model. Based on a linear relationship withthe number of amino acids, the Kd of SorC9 is expected to beapproximately 90 nM.

The TRPV6-binding peptides described herein optionally include analogsof the aforementioned peptides. Analogs of the protein of the inventionoptionally include, but are not limited to an amino acid sequencecontaining one or more amino acid substitutions, insertions, deletionsand/or mutations. Amino acid substitutions may be of a conserved ornon-conserved nature. Conserved amino acid substitutions involvereplacing one or more amino acids of the peptides of the invention withamino acids of similar charge, size, and/or hydrophobicitycharacteristics. When only conserved substitutions are made, theresulting analog should be functionally equivalent. Non-conservedsubstitutions involve replacing one or more amino acids of the aminoacid sequence with one or more amino acids that possess dissimilarcharge, size, and/or hydrophobicity characteristics. The analog isoptionally a peptoid, which is an N-substituted polyglycine with aminoacid R groups attached at the N atom. Another analog is optionally apeptide synthesized from D-amino acids rather than the natural L-aminoacids.

One or more amino acid insertions are optionally introduced into theTRPV6-binding peptide sequences of the invention. Amino acid insertionsconsist of single amino acid residues or sequential amino acids rangingfor example from 2 to 15 amino acids in length.

Deletions consist of the removal of one or more amino acids, or discreteportions from the amino acid sequence of the peptide. The deleted aminoacids may or may not be contiguous.

Analogs of a TRPV6-binding peptide of the invention are optionallyprepared by introducing mutations in a nucleotide sequence encoding thepeptide. Mutations in nucleotide sequences constructed for expression ofanalogs of a protein of the invention preserve the reading frame of thecoding sequences. Furthermore, the mutations will preferably not createcomplementary regions that could hybridize to produce secondary mRNAstructures such as loops or hairpins, which could adversely affecttranslation of the mRNA.

Mutations are optionally introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures are employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of a peptide of the invention is alsoreadily achieved by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA re-ligated. Exemplary methods of makingthe alterations set forth above are disclosed by Sambrook et al.(Sambrook J et al. 2000. Molecular Cloning: A Laboratory Manual (ThirdEdition), Cold Spring Harbor Laboratory Press).

In addition, analogs of a TRPV6-binding peptide of the invention arereadily prepared by chemical synthesis using techniques well known inthe chemistry of proteins such as solid phase synthesis (Merrifield,1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenoussolution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch,Vol. 15 I and II, Thieme, Stuttgart). The TRPV6-binding peptides of theinvention also include peptides having sequence identity to a peptide ofthe invention, mutated peptides and/or truncations thereof as describedherein. Such peptides have amino acid sequences that correspond tonucleic acid sequences that hybridize under stringent hybridizationconditions (see discussion of stringent hybridization conditions herein)with a probe used to obtain a peptide of the invention. Peptides havingsequence identity will often have the regions that are characteristic ofthe protein.

Other useful peptides of the invention optionally comprise, consistessentially of or consist of an amino acid sequence with at least: 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% sequence identity to all or part ofSEQ ID NO:1 described herein, wherein the peptide has TRPV6-bindingactivity. Sequence identity is typically assessed by the BLAST version2.1 program-advanced search (parameters as above; Altschul, S. F., Gish,W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic localalignment search tool.” J. Mol. Biol. 215:403-410). BLAST is a series ofprograms that are available online through the U.S. National Center forBiotechnology Information National Library of Medicine Building 38ABethesda, Md. 20894) The advanced Blast search is set to defaultparameters. References for the Blast Programs include: Altschul, S. F.,Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic localalignment search tool.” J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. (1993) “Identification of protein coding regions by databasesimilarity search.” Nature Genet. 3:266-272.; Madden, T. L., Tatusov, R.L. & Zhang, J. (1996) “Applications of network BLAST server” Meth.Enzymol. 266:131-141; Altschul, S. F., Madden, T. L., Schïffer, A. A.,Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs.”Nucleic Acids Res. 25:3389-3402); Zhang, J. & Madden, T. L. (1997)“PowerBLAST: A new network BLAST application for interactive orautomated sequence analysis and annotation.” Genome Res. 7:649-656). Ina further embodiment, there is provided a compound comprising anantibody to TRPV6 conjugated to a biomolecule.

Preparation of Antibodies

In one aspect, antibodies to TRPV6 are useful in accordance with theembodiments described herein. In another aspect, antibodies to theTRPV6-binding peptides, or antibodies to the compounds comprising theTRPV6-binding peptides, are useful to identify the presence of thepeptide in a test sample. Any method of labeling the antibody that wouldreport on peptide density/location would be useful (e.g. radioactivelylabeled peptide or fluorescently tagged peptide). The antibody istypically a monoclonal antibody or a polyclonal antibody. The antibodiesare also valuable for immuno-purification of peptides. For example, onemay contact a biological sample with the antibody under conditionsallowing the formation of an immunological complex between the antibodyand a peptide recognized by the antibody and detecting the presence orabsence of the immunological complex whereby the presence of the peptideof the invention is detected in the sample. The invention also includescompositions preferably including the antibody, a medium suitable forthe formation of an immunological complex between the antibody and apeptide recognized by the antibody and a reagent capable of detectingthe immunological complex to ascertain the presence of TRPV6, theTRPV6-binding peptides of the invention or similar peptides.

To recognize the TRPV6-binding peptides of the invention, one maygenerate antibodies against a range of unique epitopes throughout thepeptides. Optionally, to recognize the compounds comprising theTRPV6-binding peptides one may generate antibodies against a range ofunique epitopes throughout the compound.

Monoclonal and polyclonal antibodies are prepared according to thedescription in this application and techniques known in the art. Forexamples of methods of the preparation and uses of monoclonalantibodies, see U.S. Pat. Nos. 5,688,681, 5,688,657, 5,683,693,5,667,781, 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988,5,500,345 and 5,496,705 that are incorporated by reference in theirentirety. Examples of the preparation and uses of polyclonal antibodiesare disclosed in U.S. Pat. Nos. 5,512,282, 4,828,985, 5,225,331 and5,124,147, which are incorporated by reference in their entirety.

The term “antibody” as used herein includes fragments thereof which alsospecifically react with TRPV6 or a TRPV6-binding peptide of theinvention. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove. For example, F(ab′)2 fragments can be generated by treatingantibody with pepsin. The resulting F(ab′)2 fragment can be treated toreduce disulfide bridges to produce Fab′ fragments.

In one embodiment, a TRPV6 protein may be detected in a sample bycontacting the sample with a TRPV6-binding peptides and then detectingthe TRPV6-binding peptide with an antibody that selectively binds theTRPV6-binding peptide. In some embodiments the antibody selectivelybinds a TRPV6-binding peptide or compound of the invention but does notbind soricidin.

Compounds of TRPV6-Binding Peptide Conjugated to a Biomolecule

The embodiments described herein include novel compounds comprising aTRPV6-binding peptide conjugated to a biomolecule. As used herein, a“biomolecule” includes any atom or molecule that is detectable throughchemical, biological or physical means or exhibits chemical orbiological activity. In some embodiments, the “biomolecule” comprises aninorganic molecule such as boron clusters and iron, gold, silver andnickel nano-structures. In one embodiment, the biomolecule comprises adetectable label or a therapeutic agent. In one embodiment, thebiomolecule is a moiety of the compound that is distinguishable from theTRPV6-binding component of the compound.

As used herein, “conjugated to a biomolecule” refers to linking theTRPV6-binding peptide with a biomolecule. In some embodiments, thelinking is the result of a chemical bond between a TRPV6-binding peptideand a biomolecule. In one embodiment, the linking is a covalent bond.The TRPV6-binding peptide may also be conjugated to a biomoleculethrough the use of recombinant genetic technologies wherein a nucleicacid sequence encodes both the TRPV6-binding peptide and a proteinbiomolecule. In some embodiments, the TRPV6-binding molecule is directlylinked to a biomolecule. In other embodiments, a spacer is used to linkthe TRPV6-binding peptide with the biomolecule. In some embodiments, aTRPV6-binding molecule may also be chemically modified such that itcomprises a biomolecule, such as by radioactively labeling theTRPV6-binding peptide. In one embodiment the biomolecule is conjugatedto the TRPV6-binding peptide through a bond that can be hydrolyzed bygeneral hydrolytic enzymes.

The compounds and compositions described herein may be used to detect orbind TRPV6 protein in vivo, in vitro or ex vivo. In some embodiments,the compounds include a biomolecule that comprises a detectable label.Any suitable biomolecular labeling system known in the art may be usedto detectably label the TRPV6-binding peptides described herein. In someembodiments, the label is selected from the group consisting of aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and an enzyme. In someembodiments, the biomolecule may comprise a fluorescent, radioactive orimmunological labeled. As used herein the terms “fluorescent label” or“fluorophore” refer to a molecule or moiety that can absorb energy of aspecific wavelength and re-emit energy at a different specificwavelength. For instance, examples of fluorescent labels include, butare not limited to, Cy2, FluorX, Cy3, Cy3.5, Cy5, Cy5.5, Cy7,fluorescein isothiocyanate (FITC), Texas Red, or Rhodamine. In someembodiments, the compound includes more than one biomolecule conjugatedto TRPV6-binding peptides.

The compounds described herein can be used to deliver diagnostically ortherapeutically useful biomolecules to tumors, tissues or cells thatproduce TRPV6. In some embodiments the compound includes a biomoleculethat is detectable by Positron Emission Tomography (PET), radiometricdetection, or by Magnetic Resonance Imaging (MRI).

In one embodiment, the biomolecules conjugated to the TRPV6-bindingpeptides include metallic nano-clusters. In some embodiments, metallicclusters such as SPIO (super paramagnetic iron oxide) provide for verysensitive detection using MRI (Magnetic Resonance Imaging) that could beused for primary detection of TRPV6-rich tumors, tissues and cells. Asshown in Example 28 and FIG. 23, the TRPV6-binding peptide SorC27conjugated to SPIO targets tumors and can be used to identify cancertumors in vivo.

In some embodiments, the compounds and methods described herein allowfor the estimation of tumor volume or for following the change in tumorvolume during the course of treatment of the cancer. Microvesiclessloughed of from TRPV6-rich tumors and present in a bodily fluid arealso readily detected by metallo-TRPV6-binding peptide conjugates.

In another embodiment, the TRPV6-binding peptides are conjugated withbiomolecules that are radio-molecules such as ¹⁸F-containingbiomolecules. These compounds target TRPV6-expressing tumors, cells ortissues, and allow for the detection in vitro, ex vivo or in vivo usingPET scanning (Positron Emission Tomography) of tumors, cells or tissuesthat express TRPV6 (Cheng et al. J. Nucl. Med. 48:987-994, 2007).Similarly, microvesicles sloughed of from TRPV6-rich tumors and presentin the blood, other bodily fluids and excreta, can be detected by¹⁸F-derivatives of the TRPV6-binding peptides.

In an additional embodiment, TRPV6-binding peptides conjugated with¹²⁸I-containing biomolecules target such compounds to TRPV6-expressingtumors, cells or tissues in vitro, ex vivo or in vivo to allow fordetection using radiometric methods of these tumors, cells or tissues(Bolton et al. Biochem. J., 133, 529-539, 1973). Similarly,microvesicles sloughed off from TRPV6-rich tumors and present in theblood, other body fluids or excreta can be detected by using thecompounds described herein.

In some embodiments, the compounds disclosed herein include abiomolecule that is a therapeutic agent. As used herein, a “therapeuticagent” is a substance used in the treatment, cure, prevention orsuppression of a disease, disorder or medical condition. A therapeuticagent may also be used in the treatment, cure prevention or suppressionof any symptoms associated with a disease, disorder or medicalcondition.

In some embodiments, the therapeutic agent is an anti-cancer agent.Examples of anti-cancer agents include, but are not limited to,taxane-based drugs, platin-based drugs, anthracyclines (e.g.doxorubicin, cyclophosphamide), topoisomerase II inhibitors (e.g.etoposide), alkylating agents (e.g. ifosfamide) plant alkaloids (e.g.vinorelbine), and antimetabolites (e.g. fluorouracil).

In some embodiments, the biomolecule is a small drug molecule,oligosaccharide, antibody, antibody epitope, nanometallic cluster,radioactively-labeled molecule, taxane-based drug, anthracycline-typedrug, platin-based drug, antibiotic, anti-cancer drug, anti-fungal,anti-viral or anti-retroviral, or boron complex.

In some embodiments, the compound comprises a TRPV6-binding peptideattached to a biomolecule through a spacer. In one embodiment, thebiomolecule is a protein or peptide and the compound is a fusion proteinof the TRPV6-binding peptide and the biomolecule. Optionally, the fusionprotein includes a peptide spacer between and the TRPV6-binding peptideand the biomolecule.

The invention also includes an isolated nucleic acid encoding aTRPV6-binding peptide of the invention, such as a nucleic acid encodingSEQ ID NO:1 or one of its fragments described herein. The invention alsorelates to isolated antibodies against a peptide of the invention. Inone embodiment, the antibody optionally selectively binds a peptide ofthe invention, but does not bind to soricidin.

Compounds of TRPV6 Antibodies Conjugated to a Biomolecule

In another aspect, there are provided compounds comprising a TRPV6antibody conjugated to a biomolecule. These compounds may be generatedand used as described herein for compounds comprising TRPV6-bindingpeptides conjugated to a biomolecule. For Example, TRPV6 antibodies maybe conjugated to a biomolecule, such as a detectable label oranti-cancer agent, either directly or through a spacer.

As shown in Examples 3 and 25, antibodies to TRPV6 and the fluorescentlylabeled TRPV6-binding peptide SorC27-cy5.5 co-localize in HEK293 cellsexpressing recombinant TRPV6 as well as in samples of Grade II serouspapillary adenocarcinoma.

Detection of TRPV6

The compounds described herein bind to TRPV6 calcium channels and insome embodiments are useful to identify calcium channels in cells,tissues, tumors or microvesicles. In some embodiments, the peptides areuseful to identify cells, tumors, tissues or microvesicles that do notexpress TRPV6. In a further embodiment, the peptides are useful toidentify or label cells, tissues tumors or microvesicles that expresslarge quantities of calcium channels. In one embodiment, the compoundsdescribed herein are useful for quantifying the amount of TRPV6 in asample.

Accordingly, some embodiments described herein include a method fordetecting TRPV6 protein in a sample comprising contacting the samplewith a TRP-binding compound as described herein and detecting theTRPV6-binding compound. In one embodiment, the TRPV6-binding compoundcan be detected using an antibody that selectively binds to theTRPV6-binding peptide.

The compounds described herein that include a detectable label are alsouseful to detect TRPV6 protein or cells, tissues, tumors ormicrovesicles that express TRPV6 protein. Accordingly, embodimentsdescribed herein include methods for detecting TRPV6 protein in a samplecomprising contacting the sample with any one of the compoundscomprising a detectable biomolecule described herein and detecting thebiomolecule conjugated to the TRPV6-binding peptide.

As used herein “sample” refers to biological sample representative of anorganism or part of an organism. Examples of samples include, but arenot limited to, biological fluids, blood, tissue samples, tissuebiopsies, samples taken from tissue culture, biological fluids, tissueextracts, freshly harvested cells, microvesicles and lysates of cellswhich have been incubated in cell cultures. A “sample” may also refer toa defined area or volume of an organism, in vivo or ex vivo, such as ansample volume or area for magnetic resonance imaging. In one embodiment,the sample is an in vitro sample from a subject, such as a blood sampletaken from the subject.

As used herein, the phrase “contacting the sample” typically includes,but is not limited to, mixing or incubating a compound as describedherein with the sample, and the sample may include additional componentssuch as a buffer, solution or test reagent. “Contacting the sample” mayalso include injecting or administering a compound described herein toan organism.

Identification and Diagnosis of Cancer

TRPV6 has been shown to be overexpressed in a number of cancer celllines. The TRPV6-binding compounds disclosed herein are therefore usefulfor detecting cells that have over-expressed TRPV6 and accordingly theidentification or diagnosis of tumors or cancer in vivo, ex vivo or invitro.

The TRPV6-binding compounds described herein have been shown to bind toand co-localize with TRPV6 in vitro. As shown in FIG. 6 and Example 3, acompound comprising SorC27 conjugated to an infrared fluorescent tagbinds to TRPV6 expressed in HEK293 cells by transfection of the cellswith an expression vector, but not to HEK293 cells without a transfectedvector expressing TRPV6. FIG. 6 also shows that the TRPV6-bindingcompound co-localizes with a fluorescently labeled antibody to TRPV6.FIGS. 7-9 further show that SorC27 co-localizes with TRPV6 in prostatecancer cell line PC-3, breast cancer cell line T 47D and ovarian cancercell line SKOV-3.

TRPV6 is expressed in samples taken from human ovarian tumor biopsies.As shown in FIG. 10 and Example 4, TRPV6 transcripts were identified incDNA libraries from human ovarian tissue tumor biopsies. The amount ofTRPV6 in each sample was estimated and also quantitatively measured bydensitometry with respect to the levels for the housekeeping geneß-actin. Table 1 provides the tumor type, ratio of TRPV6/ß-actin andqualitative TRPV6 level for 18 patients with ovarian tumors.

TABLE 1 The relative amounts of TRPV6 transcripts in 18 human ovariantumor biopsies detected by PCR of cDNA libraries produced from thetumors, and a ratio of the integrated band density of the TRPV6 ampliconto that of the house keeping gene β-actin. Ratio of density PatientTRPV6/b- Qualitative Code Ovarian tumour type actin TRPV6 level LTL-175clear cell carcinoma 1.1 ++++ LTL-205 serous adenocarcinoma 0.2 ++LTL-234 mucinous carcinoma 0.8 +++ LTL-237 serous adenocarcinoma 0.4 ++LTL-246 serous carcinoma 1.8 ++++ LTL-247 serous adenocarcinoma 0.3 +++LTL-258 serous adenocarcinoma 0.5 +++ LTL-259 serous adenocarcinoma 0.4++ LTL-260 Carcinoma undifferentiated 0.5 +++ LTL-269 serousadenocarcinoma 1.8 +++++ LTL-273 endometrioid 0.6 +++ adenocarcinomaLTL-284 serous borderline 0.5 ++ LTL-290 serous carcinoma 2.9 +++LTL-300 endometrioid 1.1 +++ adenocarcinoma LTL-305 clear cell carcinoma0.3 + LTL-315 serous carcinoma 0.5 ++ LTL-317 clear cell carcinoma 0.4++ LTL-320 not known at this time 0.4 +

TRPV6 cDNA was also identified in a number of cDNA libraries preparedfrom total RNA extracts of human ovarian cancer cell lines includingOVCAR3, SKOV3, OVC13, HEYC2, and OV2008 as shown in FIG. 11.

TRPV6 expression was also determined in relation to the expression ofthe housekeeping gene ß-actin in a series of breast cancer linesincluding T47D, HCC1954 and MB468 as shown in FIGS. 12A and 12B. TheTRPV6 mRNA status results with respect to a number of breast cancer celllines and ovarian cancer cell lines is shown in Table 2:

TABLE 2 TRPV6 mRNA status of human breast and ovarian cancer cell lines.TRPV6 mRNA status Breast Cancer Cell Lines MB 231 Positive MB468Positive T 47D Positive HCC1954 Positive MCF 7 Positive Ovarian CancerCell Lines OVCar-3 Positive SKOV-3 Positive OV 90 Positive HeyC2Positive OV 2008 Positive OV C13 Positive

TRPV6 cDNA was also detected in a cDNA library made from a humanprostate cancer cell line as shown in FIG. 13.

In addition, TRPV6 protein levels were shown to be over-expressed in anumber of different cancer cell lines and ovarian tumor samples as shownin FIGS. 14-16 and Example 5.

Accordingly, embodiments disclosed herein include a method foridentifying or diagnosing cancer in a test sample from a subjectcomprising detecting TRPV6 mRNA or protein in the test sample andoptionally comparing the amount of TRPV6 mRNA or protein in the testsample with a control sample. In one embodiment, an increased amount ofTRPV6 mRNA or protein in the test sample compared to the control sampleis indicative of cancer. In one embodiment, a minimal increase of atleast 10% is indicative of cancer. In another embodiment, an increasedamount of at least: 20%, 50%, or 100% of mRNA or protein in the testsample compared to the control sample is indicative of cancer. In someembodiments, an increase of 3-times the amount of TRPV6 mRNA or proteinin the test sample compared to the control sample is indicative ofcancer. Examples of tissues where TRPV6 would be present in the controlare colon, kidney, prostate and breast. In other tissues, such as anyendothelial-derived tissues, TRPV6 is not expressed at all, so detectionof the presence of TRPV6 mRNA or protein in the test sample isindicative of the presence of cancer. Optionally, such methods areperformed on such tissues by detecting TRPV6 in the test sample, withoutalso using a control sample comparison.

TRPV6 rich tumours are nearly exclusively of epithelial origin althoughsome prostate cancer cell lines such as DU145 do not express TRPV6.Types of cancer that are TRPV6-rich include, but are not limited to,ovarian, breast, prostate, liver, endometrial, glial, colon, pancreatic,and leukemia cancers.

In some embodiments, the subject is a mammal, such as a human. In someembodiments, the methods described herein are used in vivo, ex vivo orin vitro.

As used herein, the term “control sample” includes any sample that canbe used to establish a base or normal level, and may include samplestaken from healthy persons or tissues. In some embodiments, the “controlsample” is a predetermined standardized control. In some embodiments,the “control sample” is a pre-determined value, threshold or range.

As used herein, the phrase “identifying cancer” includes the detectionof cells in a sample from a subject that have lost normal controlmechanisms and have unregulated proliferative growth. Optionally,“identifying cancer” in a sample from a subject can refer to diagnosingcancer in the subject.

In one embodiment, the methods described herein can be used to providefurther information regarding a tumor or cancer. Determination of cancerstage or type typically includes the determination of informationregarding the stage of cancer (e.g. tumor stage) or type of cancer. Inone embodiment, cancer stage is determined as known in the art using theTumor, Node, Metastasis (TNM) system. T (for Tumor) reflects on the sizeof the primary tumor and where it is located; N (for node) reflects onwhether the tumor has spread to lymph node; and, M (for Metastasis)reflects on whether the cancer has metastasized (See for example AJCCCancer Staging Manual, Seventh Edition (2010) published bySpringer-Verlag New York, herein incorporated by reference). Forexample, in one embodiment the cancer is ovarian cancer and thefollowing staging guidelines may be used:

-   -   Stage I (in ovaries): T1, N0, M0 with sub-stages I A, B, C        (where N and M remain “0”)    -   Stage II (in one or both ovaries, pelvic invasion): T2, N0, M0        with sub-stages II A, B, C (where N and M remain “0”)    -   Stage III (in ovaries, pelvic region and spread into peritoneal        area >2 cm: T3 N0, M0 with sub-stages III A, B, C (where N and M        remain “0”); Stage IIIC (into lymph): T, N1, M0    -   Stage IV (spread to distant organs): any T, any N, M1

Optionally, the methods described herein are useful to furthercharacterize a tumor by providing an estimation of tumor volume, tumorlocation, or tumor type. In some embodiments, the diagnosis of cancerincludes obtaining therapy response information such as to determine theresult of a course of anti-cancer therapy on tumor size typicallymeasured as tumor volume.

As shown in Example 24, levels of TRPV6 are higher in tissue samplesfrom subjects with cancer compared to samples of normal tissue. Morespecifically, FIG. 18 and Table 3 show that tissue micro-array samplesof Grade I, II and III serous papillary adenocarcinoma exhibited moreexpression of TRPV6 compared to samples of normal ovarian tissue. TRPV6levels are useful to distinguish between early stage Grade I cancerscompared to normal samples. Accordingly, detection of TRPV6 usingantibodies or using the TRPV6-binding peptides described herein isuseful to identify or diagnose subjects with cancer or with a greaterlikelihood of developing cancer. Optionally, levels of TRPV6 expressionare useful to grade cancers or identify more aggressive forms of cancer.

In some embodiments, methods described herein are used to identify ordiagnose breast cancer, ovarian cancer, blood cancer, brain cancer,retinal cancer, liver cancer, thyroid cancer, colon cancer, prostatecancer, pancreatic cancer, glial cancer or endometrial cancer.

In one embodiment, measuring the expression of the trpv6 gene, throughmeasurement of the amount of TRPV6 mRNA or corresponding cDNAtranscripts produced in a sample or cell line cell provides a diagnostictool with which to identify cancer in a sample. In some embodiments, thepresence or amount of TRPV6 mRNA or transcripts in a sample from asubject is used to diagnose or indicate the stage of cancer in thesubject. For example, FIG. 24 shows that the relative levels of TRPV6mRNA in blood is significantly higher in subjects with cancer comparedto levels in healthy controls. Accordingly, in one embodiment thepresence or amount of TRPV6 mRNA or transcripts in a sample from asubject is useful to identify cancer in a subject. In one embodiment,the presence or amount of TRPV6 mRNA or transcripts in a sample isuseful to identify ovarian, breast or prostate cancer in a subject.

As shown in FIG. 25, the relative amount of TRPV6 protein is also higherin samples of plasma from subjects with stage I or II ovarian cancercompared to healthy controls. Accordingly, in one embodiment thedetection of the TRPV6 protein is useful to identify cancer in asubject. For example, in one embodiment levels of TRPV6 protein in atest sample from a subject are compared to levels of TRPV6 protein in acorresponding sample from a healthy control, wherein higher levels ofTRPV6 in the test sample are indicative of cancer. In one embodiment,the test sample is a blood or plasma sample and a TRPV6 level that istwice as high as the level in a corresponding sample from a healthycontrol is indicative of cancer.

As set out in Example 1 and FIGS. 1 to 3, the TRPV6-binding peptideslocalize predominantly in lymph nodes, but also in lung, liver andkidney, which are common sites where metastatic cancer is located.Accordingly, the TRPV6-binding peptides described herein are useful fordetecting and binding to metastatic cancer, including glioblastomas andcancers that have spread to the lung, liver, kidney, spleen, pancreasand bone marrow.

The peptides and compounds described herein are particularly useful fordetecting and binding to lymph node metastases. “Lymph node metastases”optionally include lung cancer (Mujoomdar et al, 2007), gastric cancer,cervical carcinoma (Lyshchik et al., 2007), vulvar carcinoma (Vernooijet al, 2007), endometrial cancer (Aalders et al, 2007), head and necksquamous cell carcinoma (Veness et al., 2007), esophagus and throatcancer, nasopharyngeal carcinoma (Ma et al., 2007), gastrointestinalcancer (Wind et al., 2006), Gall bladder cancer, brain cancer (Mujoomdaret al., 2007), thyroid cancer, breast cancer, ovarian cancer, prostatecancer, glial cell cancer and colorectal cancer. The peptides of theinvention are therefore useful in detecting cancer in a mammal at any ofcancer stages I, II, III or IV.

The TRPV6-binding peptides and compositions described herein are alsouseful for detecting intact TRPV6 channels present in microvesiclessloughed off from tumors, circulating in the blood or cancer cellscirculating in the blood. In some embodiments, microvesicles sloughedoff from TRPV6-expressing tumors, or cancer cells circulating in bodilyfluids or excreta, are detected by PCR-based (e.g. RT-PCR or Q-RT-PCR)or antibody-based methods (e.g. Western blotting, immunofluorescentdetection in biopsies or bodies such as cells or microvesicles in bodilyfluids or excreta). For example, intact microvesicles derived from tumorcells and present in a bodily fluid or excreta would show a populationof TRPV6 channels when treated with TRPV6 antibodies or TRPV6-bindingpeptides conjugated with a detectable biomolecule. For example, FIGS. 24to 26 show that blood and plasma samples taken from subjects withcancer, including stage I cancer, have significantly higher levels ofTRPV6 mRNA or protein compared to healthy controls. Testing bodilyfluids such as blood or plasma for TRPV6 mRNA or protein thereforeprovides a relatively simple test for the early stage detection ofcancer, compared to, for example, detecting tumors and testing tumorbiopsies for the presence of cancer cells.

Antibodies developed to the TRPV6-binding peptides are useful to detectthe TRPV6-binding peptide/TRPV6 complex in tumors, tissues or cells invitro or ex vivo by tagging the TRPV6-binding peptide antibodies with adetectable entity (fluorescent tag, radioactive tag, etc.). Similarly,microvesicles sloughed of from TRPV6-rich tumors and present in bodilyfluids or excreta are readily detected by such antibodies or conjugatesof the TRPV6-binding peptides.

Drug Delivery and Method of Manufacture

The TRPV6-binding peptides and compounds described herein are useful todeliver biomolecules to tumors, cells or tissues that express TRPV6. Asshown in Examples 26 and 28, TRPV6-binding peptides are able to targetTRPV6 expressing cells and deliver compounds to tumor sites in vivo.

Accordingly, one embodiment includes a method of manufacturing apharmaceutical compound by conjugating a biomolecule to a TRPV6-bindingpeptide. Biomolecules are readily conjugated to the TRPV6-bindingpeptides or antibodies to TRPV6-binding peptides, by methods known inthe art, such as those set out in Examples 1,11-13, 16, 19-23, 26 and28.

Biomolecules can be conjugated to the TRPV6-binding peptide by linkingeither directly or indirectly the TRPV6-binding peptide to thebiomolecule. In one embodiment, the TRPV6-binding peptide is conjugatedto Cy5.5 at the single cysteine thiol through a maleamide-activatedreaction. In other embodiments, the biomolecule is liked to either theC-terminus, or N-terminus of the peptide. In other embodiments, thebiomolecule is linked through any another suitable molecular site suchas a functional group side chain of the peptide.

The biomolecule may also be conjugated to a TRPV6-binding peptide viachemical modification such as by an ester linkage or an amide linkage.Various methods of conjugating peptides to a biomolecule are disclosedfor example in Peng Li et al., Biopolymers 87: 225-230, 2007; U.S. Pat.No. 6,348,317; and U.S. Application Nos. 20070218502 and 20070020264.

In another aspect, there is provided a method of manufacturing apharmaceutical compound comprising conjugating a biomolecule to a TRPV6antibody.

Another embodiment includes a method of delivering a pharmaceuticalcomposition to a cell expressing TRPV6 comprising contacting the cellwith a compound comprising i) TRPV6-binding peptide conjugated to abiomolecule and ii) a carrier. The methods of delivering apharmaceutical composition include methods for delivering thecompositions comprising the TRPV6-binding peptides described herein.

In one embodiment, the compounds optionally comprise a TRPV6-bindingpeptide chemically altered to deliver nano-metallic clusters to tumors,tissues or cells (for example, gold nano-particles, nano-spheres,nano-tubes or other nano-constructs) that, when irradiated withelectromagnetic radiation, heat and kill cells in the vicinity of themetallic cluster.

In another embodiment, the compound comprises TRPV6-binding peptidesthat are chemically altered to deliver boron clusters (e.g. closo-boron,a cluster containing 12 boron atoms) which, when irradiated with slowthermal neutrons, produce energetic alpha particles that kill nearbycells.

Other compounds comprise TRPV6-binding peptides of the inventionchemically altered to deliver to TRPV6 producing tumors, cells ortissues, antigens that serve to recruit pre-existing antibodies to theTRPV6-rich tumors, tissues or cells. This, in turn, would mark cancersfor destruction by the immune cell system.

The compounds described herein are also useful to deliver to TRPV6producing tumors, cells or tissues, novel antigens toward whichmonoclonal antibodies are specifically developed and administered withthe result being antibody tagging of TRPV6-rich cancers. This would markcancers for destruction by the immune cell system.

The compounds described herein are also useful to deliver covalentlyattached radioactively labeled molecules to that deliver a therapeuticradiation dose to tumors, tissues or cells rich in TRPV6 channels.

The compounds described optionally deliver to TRPV6-producing tumors,cells or tissues, covalently attached therapeutics such as thetaxane-based drugs, anthracyline-type drugs, platin based drugs or anyother therapeutic molecule. The methods and compounds described hereinare also useful to deliver anti-biotics, anti-fungals, anti-virals andanti-retrovirals or any other therapeutic drug to cells that expressTRPV6 or to lymph nodes, lung, liver and/or kidney.

Detection of Cancer Tumors in a Subject

As shown in Examples 26 and 28 and corresponding FIGS. 21 and 23,detection of TRPV may be used to identify a cancer tumor in a subject invivo. As used herein “identifying a cancer tumor” refers to localizingor detecting a region in a sample or subject that has a cancer tumor. Asused herein, “cancer tumor” refers to a neoplasm or a solid lesionformed by the abnormal growth of cells that have lost normal controlmechanism and have unregulated proliferative growth. A number of cancershave been shown to overexpress TRPV6 and therefore generate TRPV6-richtumors (see Examples 4 and 5). Accordingly, in one aspect there isprovided a method for detecting a cancer tumor comprising administeringto a subject a compound comprising a TRPV6-binding peptide or anantibody to TRPV6. Preferably, the compounds also include a detectablelabel that facilitates the detection of TRPV6 in the subject. Forexample, in one embodiment the TRPV6-binding peptide is SorC27, and thedetectable label is Cy5.5. In another embodiment, the TRPV6-bindingpeptide is conjugated to a magnetic resonance imaging (MRI) contrastagent such as super-paramagnetic iron oxide and MRI is used to detectregions in a sample or subject with increased levels of TRPV6. Regionsof the subject that exhibit increased levels of TRPV6 are indicative ofa TRPV6-rich tumor in that region. Mathematical models that compare thedistribution of TRPV6 across the subject are readily applied to identifyspecific regions of the subject that have increased levels of TRPV6 thatare indicative of a TRPV6-rich tumor. In some embodiments, an averagelevel of TRPV6 observed throughout a subject is used to normalize theTRPV6 levels and identify specific regions with increased expression ofTRPV6. In other embodiments, levels of TRPV6 are compared to apre-standardized control level or to levels observed in correspondingregions in subjects known not to contain TRPV6-rich cancer tumors.

A person skilled in the art will appreciate a number of imagingtechniques and corresponding detectable labels are suitable fordetecting TRPV6-rich tumors in accordance with the present description.For example, the TRPV6-binding peptides are optionally radioactivelylabeled and detected using a scintillation counter. Alternatively, theTRPV6-binding peptides are fluorescently labeled and detected using anoptical detection system. In one embodiment, the TRPV6-binding peptidesare conjugated with a contrast agent. As used herein, a “contrast agent”is a substance used to enhance the contrast of structures or cellswithin a sample of subject in medical imaging. In one embodiment thecontrast agent is a MRI contrast agent, such as an agent that alters theT1 or T2 relaxation time of protons located nearby. Examples of MRIcontrast agents include paramagnetic gadolinium, paramagnetic manganese,or super-paramagnetic iron oxide (SPIO).

Additional Properties of the Peptides

The TRPV6-binding peptides of the compounds described herein, such asSorC13 and SorC27, are typically stable in aqueous solution at 4° C. forat least 3 weeks with no change in purity as measured by HPLC. As drysolids, the peptides are typically stable at −80° C. for at least 1.5years.

The TRPV6-binding peptides also avoid a major adverse effects ofpharmaceuticals related to the ability of a substance to cross thecentral nervous system protective barrier, the blood-brain barrier. Theinability of the peptides of the invention to cross this protectivebarrier obviates the potential toxicity to the central nervous system.

The peptides of the invention, particularly the shorter peptides, suchas SorC13, are typically less antigenic. Peptides having a number ofamino acids equal to or less than the empirical cutoff for antigenicity(typically considered to be 13 amino acids for peptides in general)possess no antigenicity.

Some embodiments include pre-packaged kits that comprise some or all ofthe reagents necessary to perform any of the methods described herein.Optionally, the kits may include one or more control samples. In someembodiments the control sample is known to express or contain TRPV6 (apositive control). In other embodiments, the control sample is anegative control that is known not to express or contain TRPV6. In afurther embodiment, the control sample is known to express or contain acertain level of TRPV6 or correspond to specific type or stage ofcancer. In some embodiments the kits include at least one compoundcomprising a TRPV6-binding peptide as described herein, and a buffersolution. In some embodiments, the kits may include nucleic acid primersfor amplifying or detecting TRPV6 mRNA in a polymerase chain reaction.In some embodiments, the kits can also include nucleotides, enzymes andbuffers useful in the method of the invention as well as electrophoreticmarkers such as a bp ladder. In some embodiments, the kits will includedetailed instructions for carrying out the methods described herein.

EXAMPLES

The following examples illustrate embodiments of the invention and donot limit the scope of the invention.

Example 1: Tissue Distribution of SorC13 and SorC27

SorC13 and SorC27 were labeled with the near-infrared probe, Cy5.5.SorC13 was labeled at lysine-1 and lysine-8 with the infraredfluorescent probe cy5.5 through reaction with Cy5.5 NHS ester-activatedprocess. SorC27 was labeled at the single cysteine thiol with Cy5.5maleimide-activated reaction. The labeled peptides were purified with acombination of size exclusion chromatography and HPLC. The label, Cy5.5,fluoresces in the infra-red region after excitation with a scanninglaser. The low energy laser is able to penetrate the animal to about 1cm and, thus, by scanning prone and supine positions, the presence ofthe tagged peptides can be quantified in three dimensions.

Cy5.5-labeled peptides were intravenously injected into CD1 mice (4 foreach compound) at 100 ug per animal in 100 uL, and animals were imagedlive using an optical imaging system, Optix eXplorer (GE HealthcareSystems) at different time points (30 min, 90 min, 4 h). Some animalswere observed at 24 hours after perfusion to remove blood (and lymph).The bio-distribution of the labeled peptides in different organs andtissues were visualized and relatively quantified by optical imaginganalysis. This protocol allows for visualization of the location of thelabeled peptides and how the location changes over time. FIG. 1 showsthe location of lymph nodes in the mouse. Nodes that accumulated thelabeled peptides are indicated by line 1 (superfacial cervical nodes),line 4 (axillary nodes), line 5 (brachial nodes), line 8 (mesentericnodes) and line 9 (inguinal nodes). FIGS. 2, 3 and 4 show the amounts oflabeled peptides in various organs ex vivo. Combined, these experimentsshow that:

-   -   Neither of the C-peptides moved across the blood-brain barrier.    -   Tagged SorC13 and SorC27 localize predominantly in lymph nodes,        lung, liver and kidney.    -   Tagged SorC13 and SorC27 were still detectable in these tissues        after perfusion at 24 hours.    -   Measurement of the fluorescence life-time in various organs        showed that metabolism of labeled peptides appears to be in        liver and kidney as Cy5.5 has a shorted life-time than        peptide/Cy5.5 adducts.    -   TRPV6-binding peptides are capable of targeting TRPV6 with a        ‘cargo’ linked to the peptides that can be delivered to these        tissues

Example 2: Co-Localization of TRPV6 Antibodies and Fluorescently-LabeledTRPV6 Expressed in Human Cancer Cells

As shown in FIG. 5, TRPV6 expressed in a cancer cell line (SKOV-3) or inex vivo samples is optionally detected using a primary antibody to theTRPV6 protein, followed by a secondary antibody that is, itself,detectable. Additionally cancers are readily detected by using theTRPV6-binding peptides conjugated or tagged with a detectable molecule.Alternatively, an antibody developed to the TRPV6-binding peptides isreadily tagged with a detectable entity such as a fluorescent tag orradioactive tag.

As well, an antibody to the TRPV6-binding peptides is readily developedand used in traditional immunochemical fashion for tissues (in vitro),tumors, cells or microvesicles.

Example 3: Co-Localization of Fluorescently-Labeled TRPV6-BindingPeptides and Antibodies to TRPV6 in HEK269 Cells

HEK-293 cells were transfected with a TRPV6 expression vector andincubated with fluorescently labeled antibodies to the N-terminalpeptides of TRPV6 as well as fluorescently labeled SorC27(SorC27-cy5.5). HEK-293 cells that were not transfected with a TRPV6expression vector were also incubated with fluorescently labeledanti-TRPV6 and SorC27-cy5.5 as a negative control (FIG. 6A).

As shown in FIGS. 6B to 6D, SorC27-cy5.5 bound to TRPV6 as indicated bythe co-localization of SorC27-cy5.5 and anti-TRPV6. This experimentshows that the compounds described herein target and bind to TRPV6 andfurther that compounds comprising a TRPV6-binding peptide conjugated toa biomolecule, are effectively localized to cells that express TRPV6.

Example 4: Expression of TRPV6 mRNA in Cancer Cell Lines and TumorBiopsies as a Diagnostic for Cancer and for Staging Cancers

As shown in FIGS. 10-13, polymerase chain reaction with primers directedtowards TRPV6 transcripts detects up-regulation of TRPV6 mRNA in cDNAlibraries produced from extracts of total RNA of cancer cells orbiopsies of human cancerous tumors.

In order to quantify the relative amount of TRPV6 transcripts in asample, transcripts were also amplified and detected for thehousekeeping gene ß-actin and the observed ratio of TRPV6 ß-actin wasrecorded.

A common mechanism is in effect in all of the cancer types referencedherein, as they are all derived from epithelial tissues. Peng et al.showed in prostate cancer that normalized amounts of TRPV6 mRNA wereabout 2-fold greater in samples from subjects with Gleason scores of 5-7as compared to samples with benign hyperplasia, and about 3-fold greaterin samples with Gleason scores of 8-9.

FIGS. 10-13 show a significant increase in the expression of TRPV6 insamples of cancer lines such as ovarian cancer. The methods describedherein that detect either TRPV6 mRNA or protein levels are thereforeuseful for the staging of cancers such as ovarian cancer.

Example 5: Analysis of TRPV6 Protein in Cancer Cells

Western blotting using antibodies developed to TRPV6 was used to detectthe expression of TRPV6 protein in a number of cancer cells.

As shown in FIG. 14, TRPV6 protein was over-expressed in extracts fromovarian (SKOV-3), breast (T47D) and prostate cancer (PC-3) cell linescompared to extracts from a hepatoblastoma HEP G2 control cell lineknown to express TRPV6.

FIGS. 15A through 15D show that extracts from human ovarian tumors alsooverexpress TRPV6 protein and that TRPV6 is upregulated in ovariantumors.

FIG. 16 shows that TRPV6 is over-expressed in extracts from humanglioblastoma (U87MG), human colon (CaCo-2) and pancreatic carcinomacells (Panc1). Further, the degree of de-glycosylation in Panc1increases with increase numbers of culture passaging.

Collectively the data in FIGS. 14 to 16 shows that TRPV6 protein isoverexpressed in a number of cancer cells and ovarian tumor samples.

Example 6: Use of TRPV6 Antibodies to Determine Cancer Stages

Antibodies to TRPV6 are used to determine the stage of a tumor (e.g.ovarian tumors) using immunolocalization methods. Staged tissuemicroarrays of ovarian tumors are probed with fluorescentlylabeled-TRPV6 antibodies.

Results

Experiments show that, as the stage of the tumor progresses throughStage I to Stage IV there is an increase in the density of theimmunofluorescence signal indicating an increase in the population ofTRPV6 channels in the tissues.

Example 7: Use of Fluorescently Tagged TRPV6-Binding Peptides toDetermine Cancer Stages

Fluorescently tagged TRPV6-binding peptides are useful to determine thestage of a tumor by direct incubation of Tissue Micro-Arrays (TMA) withthe peptide reagent. SorC27-cy5.5 compound is incubated with cells froma subject and fluorescence levels are compared to levels from controlstaged tumor samples.

Results

Experiments show that, as the stage of the tumor progresses throughStage I to Stage IV, there is an increase in the intensity of thefluorescent signal due to binding of the tagged compound comprisingSorC27-tagged with cy5.5 to the increased population of TRPV6 channels.

Example 8: Use of Fluorescently Tagged Antibodies to CompoundsComprising TRPV6-Binding Peptides to Determine Cancer Stages

Fluorescently tagged antibodies to TRPV6-binding peptides are used todetect TRPV6-binding peptides bound to TRPV6 channels of a validatedovarian cancer tissue microarray.

Results

After incubating the TMA with one of the TRPV6-binding peptides, andthen deploying a fluorescently tagged antibody developed against theTRPV6-binding peptides, there is a positive correlation between theintensity of the fluorescent signal with the stage of the cancer fromStage I through Stage IV.

Example 9: Fluorescently-Labeled TRPV6-Binding Peptides Bind toTRPV6-Rich Tumors Xenografted in Mice

Mice xenografted with an ovarian TRPV6-rich tumor (SKOV-3 cells) areinjected with the fluorescently-labeled TRPV6-binding peptideSorC27-cy5.5.

Results

The ovarian tumors over-expressing the TRPV6 channel are detected invivo by SorC27-cy5.5 compound after administration to mice in whichTRPV6 tumors are xenografted (e.g. ovarian tumors). The TRPV6-richxenografted tumor mass derived from SKOV-3 cells is clearlydistinguished from the background tissue as strongly fluorescing in thefar infrared.

Example 10: Use of Super Paramagnetic Iron Oxide-Labeled (SPIO)TRPV6-Binding Peptides to Detect TRPV6-Producing Cells

A compound comprising TRPV6-binding peptide conjugated with SuperParamagnetic Iron Oxide such as SPIO-SorC27 is incubated with SKOV-3cells (TRPV6 positive) and HEK293 cells (TRPV6 negative). The cells arethen imaged using Magnetic Resonance Imaging.

Results

SKOV-3 cells over-expressing the TRPV6 channel are detected in vitro byMRI enhancement agents such as SPIO-SorC27. The TRPV6-rich SKOV-3 cellsare clearly distinguishable from the TRPV6-negative HEK293 cells bystrongly enhanced magnetic resonance signals and imaging.

Example 11: SPIO-Labeled TRPV6-Binding Peptides Bind to TRPV6-ExpressingTumors but not to TRPV6-Negative Tumors

Mice xenografted with a SKOV-3 TRPV6-rich ovarian tumor are administeredSPIO-SorC27 and imaged using Magnetic Resonance Imaging.

Results

The results show SPIO-conjugated TRPV6-binding peptides bind toTRPV6-rich tumors from human ovarian cancer cell line SKOV-3, and canimaged using Magnetic Resonance Imaging. Experiments show that ovariantumors over-expressing the TRPV6 channel are detected in vivo by MRIenhancement agents such as SPIO-SorC27 after administration to themouse. The TRPV6-rich xenografted tumor mass is clearly distinguishedfrom the background tissue by strongly enhanced magnetic resonancesignals and imaging.

Example 12: Use TRPV6-Binding Peptides Conjugated to ¹⁸F-ContainingRadio-Molecules

Compounds comprising TRPV6-binding peptides covalently labeled with¹⁸F-containing radio-molecules target such molecules to TRPV6-expressingtumors, cells or tissues and allow for detection using PET scanning(Positron Emission Tomography) and detection of these tumors, cells ortissues (Cheng et al. J. Nucl. Med. 48:987-994, 2007). SorC27 conjugatedwith an ¹⁸F-containing radio-molecule is incubated with SKOV-3 cells andHEK293 cells. The cells are then imaged using PET scanning.

Results

The ¹⁸F-SorC27 clearly shows the identification of the TRPV6-rich SKOV-3cells as compared to TRPV6-negative cells (HEK293).

Example 13: Use TRPV6-Binding Peptides Conjugated to ¹²⁵I-ContainingRadio-Molecules

Compounds comprising TRPV6-binding peptides covalently labeled with¹²⁵I-containing radio-molecules target such molecules toTRPV6-expressing tumors, cells or tissues and allow for detection usingradiometric detection of these tumors, cells or tissues (Bolton et al.Biochem. J., 133, 529-539, 1973). SorC27 conjugated with a¹²⁵I-containing radio-molecule is incubated with SKOV-3 cells and HEK293cells. The cells are then imaged using PET scanning.

Results

The ¹²⁵I-C-peptide clearly shows the identification of the TRPV6-richSKOV-3 cells as compared to TRPV6-negative cells (HEK293).

Example 14: Detection of TRPV6 mRNA in Microvesicles Isolated fromSamples of Blood, Other Body Fluids or Excreta

TRPV6 mRNA is detected in microvesicles isolated from samples of blood,other body fluids or excreta of patients with TRPV6-rich cancers (e.g.ovarian tumors). The fraction of blood containing microvesicles sloughedoff of tumors through an endocytotic process is isolated from a sampleobtained from a subject. RNA is then extracted from these microvesicles,with subsequent analysis by PCR-based techniques.

Results

Q-RT-PCR shows the presence of TRPV6 mRNA at excess in cancer patientblood samples in comparison to people without a TRPV6-rich cancer.Detection of relative amounts of TRPV6 expression is therefore useful todiagnose TRPV6-rich cancers such as ovarian cancer.

Example 15: Detection of TRPV6 Protein in Microvesicles Isolated fromBodily Fluids or Excreta

TRPV6 protein is detected in microvesicles isolated from bodily fluidssuch as blood, lymph or excreta samples of patients with TRPV6-richcancers (e.g. ovarian tumors). The fraction of the sample containingmicrovesicles sloughed off of tumors through an endocytotic process isisolated. Protein is then extracted from these microvesicles, withsubsequent analysis by Western blotting and antibody-based techniques todetect TRPV6 protein. Alternatively, isolated microvesicles can betreated with either anti-TRPV6 antibodies or TRPV6-binding peptides anddetected.

Results

Western blotting shows the presence of TRPV6 protein at excess in cancerpatient blood samples in comparison to people without a TRPV6-richcancer such as ovarian cancer. Whole microvesicles show TRPV6 byimmunofluorescence using an antibody to TRPV6 and by using appropriatelylabeled TRPV6-binding peptides.

Example 16: In Vivo Use of TRPV6-Binding Peptides Conjugated to Taxanes

It is clear from the bio-distribution studies set out in Example 1 thatthe compounds comprising TRPV6-binding peptides are useful to deliverthe attached fluorophore of the cyanidine class (cy5.5) to tissues, andto cells expressing TRPV6 (See FIGS. 6 to 9). Compounds comprising theTRPV6-binding peptides described herein may therefore be used to deliverother molecules to cells or tissues expressing TRPV6.

Compounds are prepared by covalent attachment of oncology drugs such asa taxanes to the TRPV6-binding peptides in order to use theTRPV6-targetting function of the peptides to deliver the drug directlyto a TRPV6-rich tumor or cancer cell. The chemistry for attaching suchdrugs to compounds is known in the art (see for example, Peng Li et al.,Biopolymers 87: 225-230, 2007).

Paclitaxel is attached through a four carbon spacer molecule to theSorC27 peptide at a primary amine or free thiol group. TheSorC27-conjugated taxane is then administered to mice xenografted with aTRPV6-rich tumor. Control mice are administered saline solution.

Results

In vivo experiments in mice show that a compound comprising aTRPV6-binding peptide conjugated to paclitaxel results in the adequatedelivery of paclitaxel to the tumor site, regression of the tumor andthe death of cancer cells when compared to control mice administeredsaline solution.

Example 17: Delivery of Anti-Viral Drugs Conjugated to TRPV6-BindingPeptides

Anti-viral or anti-retroviral drugs are readily covalently attached toTRPV6-binding peptides for the treatment of reservoirs of HIV. A primaryreservoir of HIV in humans is the mesenteric lymph nodes (Cumont et al.Cell Death and Differentiation, 14, 1747-1758, 2007; Estaquier andHurtrel. Medical Science (Paris) 24(12): 1055-1060, 2008). Compoundscomprising the TRPV6-binding peptides carrying a fluorescent tagaccumulate in the mesenteric lymph nodes as shown by the presence offluorescence in these nodes after i.v. injection of the tagged peptide(see FIG. 1).

Anti-viral or anti-retrovirals are covalently attached to theTRPV6-binding peptides described herein. A compound comprising aSorC27-anti-viral conjugate is then administered to mice following theprotocol set out in Example 1.

Results

The results show the anti-viral conjugate is detected in lymph nodetissue. Anti-viral activity is obtained, blocking HIV reproduction andthereby treating HIV.

Example 18: TRPV6-Binding Peptides Conjugated to Anti-Microbials

The covalent attachment of anti-microbial drugs to the TRPV6-bindingpeptides is useful for the treatment of reservoirs of bacterialinfection. Compounds comprising the TRPV6-binding peptide conjugated toa fluorescent tag accumulate in the lymph nodes as indicated by thepresence of fluorescence in these nodes after iv injection of the taggedpeptide (see FIG. 1). Similarly, anti-microbials are optionallycovalently attached to the TRPV6-binding peptides by methods known inthe art, and used to target lymph nodes.

Results

The results show that the drugs are detected in lymph node tissueisolated from mice treated with the anti-microbial-TRPV6-bindingconjugates and will kill microbes.

Example 19: TRPV6-Binding Peptides Conjugated to Boron Complexes

Compounds comprising a boron complex covalently attached to aTRPV6-binding peptide are useful for cancer therapy. The TRPV6-bindingpeptides target and contact TRPV6-rich cells and tumors causingconcentration of the boron complexes therein (each with multiple boronatoms, for example the 12 boron closo-boron complex). Irradiation withthermal neutrons results in neutron capture by boron-10 atoms andtransfer of high energy alpha-particles to the tissue killing tumorcells. This aspect of the invention provides for large amounts of boronclusters being targeted to tumors. The threshold has been stated to beabout 20 ug B/g tumor (Barth et al. Clinical Cancer Research, 11 (11)3987-4002, 2005). The chemistry to attach boron compounds to proteincomplexes is a well established (Guan et al. Proc. Natl. Acad. Sci., 9513206-13210, 1998). Boron conjugated to TRPV6-binding peptides isadministered to mice xenografted with a TRPV6-rich tumor. The tumor isthen irradiated with thermal neutrons.

Results

The concentration of boron is greatly increased in TRPV6-rich tumorssuch as ovarian tumors. Irradiation of the tumor with thermal neutronskills the tumor cells. The boron-peptide compounds are useful forkilling tumor cells.

Example 20: TRPV6-Binding Peptides Conjugated to Epitopes

The covalent attachment of known and/or recognized epitopes of globalantibodies to the TRPV6-binding peptides recruit these preexistingantibodies to TRPV6-expressing tumors and cancer cells. This complex(antibody-epitope-TRPV6-binding peptide) results in detection anddestruction of TRPV6-expressing cells by immune cells as shown inxenograft experiments where tumors (e.g. ovarian) had lower growthrates.

Example 21: TRPV6-Binding Peptides Conjugated to Antigens or Epitopes

The covalent attachment of a novel antigen or epitope to theTRPV6-binding peptides and administration of the compound to micedirects the epitope/TRPV6-binding peptide complex to the TRPV6-bearingtumors. Subsequent administration of a monoclonal or polyclonal antibodydirected against the specific epitope results in either type of antibodyattaching to the TPRV6-rich cell, tissue or tumor. Subsequentrecruitment of immune cells (e.g. killer T-cells) results in death ofthese peptide-targeted cells and shrinkage of the tumors.

Example 22: TRPV6-Binding Peptides Conjugated to Immunoactivators

The covalent attachment of TRPV6-binding peptides and a molecule that isrecognized and bound by receptors on cells of the immune system,particularly killer T-cells, directs such cells to TRPV6-rich tumors orcancer cells. The recruitment of such cells as killer T-cells destroythe cancer cell or tumor. The experiments indicate that theTRPV6-binding peptide targets the TRPV6 channel while the ‘batemolecule’ attached to the other end of the TRPV6-binding peptidesrecruits the immune cell; administration of this product causesshrinkage of tumors (e.g. xenografted ovarian tumors).

Example 23: TRPV6-Binding Peptides Conjugated to MetallicNano-Structures

The covalent attachment of metallic nano-structures (e.g nano-goldparticles) to the TRPV6-binding peptides targets the metalnano-particles or other constructs (spheres, rods etc.) through theTRPV6 binding function of the peptides, to cancer cells and tumors.Because of the unique properties of, for example, nano-gold particles,irradiation with radio frequencies or infrared radiation cause theparticles to heat up. Subsequent heating of the cancer cell or tumorcauses death of the cells or tumor. The chemistry to attach metallicnano-particles to molecules such as peptides, is well established.Experiments indicate that targeting of the nano-gold conjugate, toTRPV6-rich xenografted tumors (e.g. ovarian tumors), with subsequentirradiation causes shrinkage of the tumor mass.

Example 24: Use of TRPV6 Antibodies to Detect and Grade Cancer

Samples comprising tissue sections of 146 independent biopsies over 4tissue micro-arrays (TMA) (OV483, OV802, T112, BCN721 obtained from USBiomax, Inc. Rockville, Md. 20850, USA) were tested forimmunohistochemical staining of TRPV6 using a TRPV6 antibody, secondaryantibody and colorimetric detection using horseradish peroxidase (HRP).The samples included eighteen different ovarian cancer typesrepresenting all the major types of ovarian cancers as well as 21samples of normal ovarian tissues. Many of the samples representedcancers that were previously graded. FIG. 17 shows the calibration andrepresentative grading of TMA samples on a six-point scale from (−) to(++++) used to assess intensity of TRPV6 staining. As shown in Table 3,each of the 146 samples was ranked according to this six-point scale.

FIG. 18 shows that a much smaller percentage of normal ovarian tissuesamples had a TRPV6 stain intensity score ≥1+ compared to serouspapillary adenocarcinoma tissue samples with grade I, grade II, or gradeIII cancer. For example, 100% of serous papillary adenocarcinoma tissuesamples had a stain intensity score of 1, compared with only about 24%of normal ovarian tissues. Accordingly, detection of TRPV6 such as byantibody staining is useful in order to predict or diagnose thelikelihood of cancer in a sample. Furthermore, detection of TRPV6 isuseful to identify samples with Grade I (early stage) cancer compared tonormal ovarian tissue samples.

FIG. 19 shows examples of the immunohistochemical detection of TRPV6using TRPV6 antibodies in micro-array samples of normal ovarian tissues,as well as samples of Grades I, II and III serous papillary carcinoma.FIG. 19 shows an observed trend of increased staining of TRPV6 insamples with higher Grades of cancer.

Cancer staging data was also available for a number of tissue arraysamples. Cancers were staged according to the Tumor, Node, Metastasis(TNM) system as known in the art. TRPV6 immunohistochemical stainingintensity for each sample assessed on a five-point scale from (−) to(++++) using a TRPV6 antibody, secondary antibody and colorimetricdetection using horseradish peroxidase (HRP) is provided in Table 4.

As shown in Table 4, only 23.8% (5/21) of normal ovarian tissue sampleshad a TRPV6 stain intensity score greater than or equal to +1. Incontrast, 95.7% (44/46) of samples with stage I-IV cancer had a TRPV6intensity score greater than or equal to +1. Early stage cancers (stageI and stage II) were readily detected with 92.9% (26/28) of early stagecancers having a TRPV6 stain intensity score greater than or equal to+1. Detection of TRPV6, such as by immunohistochemical methods, cantherefore be used to detect cancer and in particular early stagecancers.

TABLE 3 Ovarian carcinoma and normal tissue array TRPV6 staining in 146samples. Total TRPV6 IHC Results Case (Stain Intensity) PathologyDiagnosis Number − −/+ + ++ +++ ++++ Normal ovarian tissue 21 8 8 3  2 0  0 Mucinous papillary 12 1 0 3  0  4  4 adenocarcinoma Total Grade I 2 0 0 0  0  1  1 Grade II  7 1 0 1  0  2  3 Grade III  3 0 0 2  0  1  0Mucinous papillary 14 2 0 2  4  4  2 cystadenocarcinoma Total Grade I 112 0 2  3  4  0 Grade II  3 0 0 0  1  0  2 Serous papillary 58 0 0 7 1318 20 adenocarcinoma Total Grade I  8 0 0 2  2  0  4 Grade II 22 0 0 1 6  8  7 Grade III 28 0 0 4  5 10  9 Serous papillary  3 0 0 0  1  2  0cystadenocarcinoma Total (Grade III) Clear cell carcinoma  9 0 0 1  6  2 0 Endometroid adenocarcinoma  4 0 0 0  0  3  1 Transitional cellcarcinoma  1 0 0 0  0  1  0 Granular cell tumor  3 0 1 2  0  0  0Endodermal sinus carcinoma  2 0 0 0  0  0  2 Squamous cell carcinoma  10 0 0  0  1  0 Metastatic signet-ring cell  3 0 0 1  1  1  0 carcinomaMetastatic adenocarcinoma  6 0 0 0  0  2  4 Mixed germ cell tumor  1 0 00  0  1  0 Malignant follicular theca  1 0 0 0  1  0  0 cytoma Malignanttumor (sparse)  2 0 0 0  2  0  0 Dysgerminoma  3 0 0 0  2  1  0 Immatureteratoma  1 0 0 1  0  0  0 Hyperplastic fibrous tissue  1 0 0 1  0  0  0

TABLE 4 Ovarian carcinoma and normal tissue arrays grouped by cancerstage. Total number of cases = 67; total number of tumor samples = 46.Total TRPV6 IHC Results Case (Stain Intensity) Pathology DiagnosisNumber − + ++ +++ ++++ Normal ovarian tissue 21 16 3 2 0 0 Mucinouspapillary 11  1 3 0 3 4 adenocarcinoma Total Stage I  5  0 0 0 2 3 StageII  1  1 0 0 0 0 Stage III  3  0 1 0 1 1 Stage IV  2  0 2 0 0 0 Mucinouspapillary 12  1 2 4 4 1 cystadenocarcinoma Total Stage I  9  1 1 3 3 1Stage II  1  0 0 0 1 0 Stage III  1  0 1 0 0 0 Stage IV  1  0 0 1 0 0Serous papillary 15  0 2 5 3 5 adenocarcinoma Total  7  0 1 3 0 3 StageII  2  0 0 0 1 1 Stage III  6  0 1 2 2 1 Stage IV  0  0 0 0 0 0 Clearcell carcinoma  3  0 2 1 0 0 (Stage I) Endometroid  4  0 0 0 3 1adenocarcinoma Transitional cell  1  0 0 0 1 0 carcinoma

Example 25: Co-Localization of TRPV6 and Fluorescently-Labeled Sorc27 inGrade II Serous Papillary Adenocarcinoma

To test the efficacy of tagged SorC27 peptide to detect its bindingtarget TRPV6, standard immunohistochemical and SorC27-cy5.5 bindingprotocols were applied to tissue micro-arrays of a number of ovariancancer tumor sections. The detection of TRPV6 channel by both methodswas observed across different ovarian cancer microarray samples.

Results

FIG. 20 shows TRPV6 antibody detection and fluorescent detection atissue micro-array sample of grade II serous papillary adenocarcinomastained with antibodies to TRPV6 as well as SorC27 fluorescently labeledwith cy5.5 (SorC27-cy5.5). The use of fluorescently-labeledTRPV6-binding peptides therefore corresponds to the standardimmunohistochemical detection of TRPV6 ion channels and TRPV6-bindingpeptides are useful to reliably detect TRPV6 or cells or tissues thatexpress TRPV6.

Example 26: Localization of SorC27 to Xenografted Cancer Tumors In Vivo

Mice xenografted with a TRPV6-rich ovarian (SKOV-3 cells) and prostate(DU145 cells) tumors were injected (intraperitoneally) with thefluorescently-labeled TRPV6-binding peptide SorC27-cy5.5. SorC27 waslabeled with the near-infrared probe Cy5.5 at the single cysteine thiolusing a Cy5.5 maleimide-activated reaction. The labeled peptides werepurified with a combination of size exclusion chromatography and HPLC.Cy5.5, fluoresces in the infra-red region after excitation with ascanning laser. The low energy laser is able to penetrate the animal toabout 1 cm and, thus, by scanning, the presence of the tagged peptidescan be quantified in three dimensions. Cy5.5-labeled peptides wereintraperitoneally injected into nude CD1 mice at 100 ug per animal, andanimals were imaged live using an optical imaging system, Optix eXplorer(GE Healthcare Systems) at different time points (30 min, 90 min, 4 h).

Results

The ovarian and prostate tumors over-expressing the TRPV6 channel areclearly detected in vivo for at least 24 hours after injection. TheTRPV6-rich xenografted tumor masses are clearly distinguished from thebackground tissue as strongly fluorescing in the far infrared. FIG. 21Ashows the time dependent localization of SorC27-cy5.5 in ovarian tumor,while FIG. 21B shows the time dependent localization of SorC27-cy5.5 inprostate tumor. The kidneys are also highlighted in these two images.The 3-D nature of the scanning device allows clear discriminationbetween the tumors and the visible kidney tissues by isolating a 2 mmslice of the animal. As well, a ‘slice’ through the tumor (the Z-slice)allows clear observation of the fluorescing central region of thetumors.

Example 27: Quantitative RT-PCR of TRPV6 mRNA in Cancer Tissues

Quantitative RT-PCR for TRPV6 mRNA was performed on samples of ovarian,prostate and breast cancer biopsies as well as corresponding pooledcontrol samples from 15 healthy individuals. 18 ovarian tumor biopsies,4 prostate tumor biopsies, and 3 breast tumor biopsies were tested. 3different RT-PCR primer sets (A, B and C) were used for testing of theprostate samples. Results were standardized against the expressionlevels of the housekeeping gene hypoxanthine phosphoribosyl transferase(HPRT) and expressed as a ratio of the standardized signal from thetumor samples to the standardized signals from a pooled sample of 15healthy tissues.

Results

FIG. 22 shows the results of Q-RT-PCR quantification of TRPV6 mRNAextracted from human ovarian (A), prostate (B) and breast cancerbiopsies (C), compared to healthy tissues. Tables 5 to 7 also providethe quantitative Q-RT-PCR results for each of the sample biopsiesrelative to normal controls. Tumor biopsies showed a significantincrease in expression levels of TRPV6 mRNA. Increases in TRPV6 mRNAexpression relative to control tissues were seen in each cancer sampletested except one ovarian cancer sample (LTL290). Ovarian cancer samplesshowed an average 39 times increase in the expression of TRPV6 mRNAcompared to healthy controls, while prostate cancer samples and breastcancer samples exhibited 8.7 times and 13 times increases respectively.The significant increases observed in transcription of TRPV6 mRNA incancer tissues provides a useful diagnostic or prognostic tool foridentifying cancer including ovarian, breast and/or prostate cancers.

TABLE 5 TRPV6 mRNA quantitative RT-PCR results for ovarian cancerbiopsies. Average Relative Increase in TRPV6 Sample ID mRNA ExpressionS.D n LTL175 2.93 0.6 3 LTL205 10.08 1.7 3 LTL234 12.04 1.0 3 LTL23717.62 3.3 3 LTL246 5.94 0.0 3 LTL247 41.52 3.0 3 LTL258 4.54 0.1 3LTL259 4.55 1.4 3 LTL260 4.87 0.4 3 LTL269 14.56 0.6 3 LTL273 12.44 0.33 LTL284 13.49 1.6 3 LTL290 0.44 0.0 3 LTL300 101.15 9.1 3 LTL305 13.423.0 3 LTL315 72.90 7.0 3 LTL317 20.23 1.2 3 LTL320 354.02 33.8 3 Average39.3 Median 12.9 S.D. = standard deviation; n = number of samples.

TABLE 6 TRPV6 mRNA quantitative RT-PCR results for prostate cancerbiopsies. Relative Increase in TRPV6 mRNA Expression Sample ID PrimerSet A Primer Set B Primer Set C A5 12.3 4.9 5.4 A11 20.8 5.9 9.6 A12 9.12.4 5.6 PA-T 16.3 3.1 Average 8.7 Median 5.9 Average and median takenfor all primer sets across each sample.

TABLE 7 TRPV6 mRNA quantitative RT-PCR results for breast cancerbiopsies. Sample ID Relative Increase in TRPV6 mRNA Expression FBT-122.5 FBT-2 3.6 FBT-3 12.9 Average 13

Example 28: In Vivo Injection and MRI Imaging of TRPV6-Binding PeptideConjugates (SPIO-SorC27) Conjugation of SorC27 to SPIO Nano-Beads

SPIO (Super Paramagnetic Iron Oxide) beads functionalized withapproximately 120 maleimide groups per bead (Product No. 77-96-201 frommicromod Partikeltechnologie GmbH, Germany) were reacted with a 5-foldmolar excess of buffered Sor-C27 (1 mM, 10×PBS, pH 7.2) for 1 hour atroom temperature. The beads were separated from the reaction mixture bycentrifugation and suspended in a volume of sterile Dulbecco's PBS forinjection into the tumor-bearing CD-1 nude mice. The number of SOR-C27peptides per bead was determined by quantitative ¹H NMR analysis of thesupernatant to determine number of reacted peptide molecules. On average75 molecules of SOR-C27 were conjugated to each SPIO particle.Conjugation of the peptide to the SPIO was confirmed by LC-MS aftertrypsin digest of the SPIO-peptide conjugate. The SPIO-SorC27 conjugatewas then injected intraperitoneally (i.p.) into SKOV-3 derived ovariantumors xenografted into CD-1 nude mice prior to imaging.

MRI Image Capture

The MRI images were acquired on a 3T Varian Direct Drive Console using a305/210 mm OD/ID Magnex gradient coil and a 25 mm diameter quadraturemouse RF coil from Doty Inc. The images were acquired using a pulsesequence specifically selected to optimize contrast sensitivity toiron-oxide nanoparticles. The iron-oxide appears dark (negativecontrast) for these types of acquisitions. The acquisitions used a 3Dbalanced steady state free precession (b-SSFP) pulse sequence with aTR/TE of 8/4 ms and an image resolution of 150 micron (150 micron pixeldimension in all 3 directions).

Results

FIG. 23 shows MRI images and the localization of the MRI enhancementagent (SPIO-SorC27) to SKOV-3 derived ovarian tumors xenografted intoCD-1 nude mice. The upper control panels (A) show the administration ofthe SPIO control beads, without conjugated SorC27. The SPIO controlbeads were cleared from the tumor by 24 hours post-injection. The lowerlevel panel (B) shows that the SPIO-SorC27 compound labels the cortex ofthe tumor 24 hours post-injection. The solid white arrow shown in theleft-hand images indicates the position of the tumor in the xenograft.The dashed arrow in the bottom right panel indicates the darkenedenhanced MRI signal of the SPIO-SorC27 construct bound to the cortex ofthe tumor. No corresponding accumulation of the iron nano-particles isobserved in the top right control panel. The panels on the right handside show MRI imaging before i.p. injection, while those on theright-hand side show MRI imaging 24 hours after administration of thecontrol or diagnostic reagent. Conjugated TRPV6-binding peptides such asSorC27-conjugates are therefore able to effectively target tumor sitesin vivo.

Example 29: RT-PCR of Blood from Staged Cancer Subjects

Samples of blood taken from subjects with prostate, breast or ovariancancer (stages I to IV) were tested for TRPV6 mRNA expression usingRT-PCR. Control samples of blood taken from a healthy male (prostate) orhealthy female (breast and ovarian) were also tested. RT-PCR productswere loaded onto agarose gels and separated using electrophoresis.Integrated band density was then measured on the agarose gels for theamplicons of the TRPV6 mRNA (˜320 bp) for each sample and control.

Results

FIG. 24 shows that the expression of TRPV6 mRNA in blood samples takenfrom patients with cancer was significantly higher compared to samplestaken from normal healthy controls. FIG. 24A shows that subjects withstage I, II, III or IV prostate cancer had up to 6 times more expressionof TRPV6 in blood. A significant increase in TRPV6 expression is alsoseen in samples representing different cancer stages compared to normalsamples. FIG. 24B shows that subjects with breast cancer exhibit asignificant increase in blood TRPV6 expression. FIG. 24C shows thatsubjects with ovarian cancer also exhibit an increase in TRPV6expression compared to healthy female control samples. The integratedsignals represented in FIG. 24 were divided by a factor of either100,000 (24A & 24B) or 10,000 (24C). Analysis of expression levels ofTRPV6 mRNA in blood is therefore useful to identify subjects withcancer, including the early detection of stage I prostate, breast orovarian cancer.

Example 30: TRPV6 mRNA in Plasma Samples from Subjects with Stage I orStage II Ovarian Cancer

Total RNA was extracted from plasma samples from healthy women (10) andfrom women with Stage I (3) or Stage II (3) ovarian cancer using the TRIReagent® LS method (Sigma Aldrich). After preparation of cDNA libraries(iScript, BioRad) from an equal amount of the extracted RNA from eachsample, the samples were subjected to standard PCR. PCR reactions wereanalyzed using E-Gel® EX 1% agarose gels and run on the EGel® iBase gelelectrophoresis unit. Using Program 7, for E-Gel® EX 1-2% gels, for 10mins. The TRPV6 mRNA (cDNA) amplicons were imaged and quantified with anAlpha Innotech FluorChem® FC2 imager, using filter position #1 (greenfilter) and UV light (302 nm) using Auto Expose. All samples wereanalyzed in triplicate and data were compared using the Student's t-testand the 95% confidence limit.

Results

As shown in FIG. 25, plasma taken from subjects with stage I or IIovarian cancer had significantly more expression of TRPV6 mRNA (p<0.0001OVI; p=0.0475 OVII) compared samples from healthy controls.

Example 31: Analysis of TRPV6 Protein Levels in Subjects with Stage Iand II Ovarian Cancer

Plasma samples were obtained from healthy women and women diagnosed withstage I or II ovarian cancer. Protein was isolated from the plasmasamples following the TRI Reagent® LS method (Sigma Aldrich). Lysateswere prepared from the plasma protein pellets from the TRI Reagent® LSprocedure by heating in a solution of 1% SDS in PBS and 15 mMdithiothrietol (DTT) in a boiling water bath. Protein extracts werequantified by measuring the absorbance at 280 nm of each lysate on aVarian Cary 50 UV spectrophotometer. The amount of protein in ug/uL wasextrapolated from a bovine serum albumin protein standard curve. Proteinextracts were electrophoresed on NuPage® Novex 4-12% Bis-Tris Gel 1.5 mmwells (Invitrogen) at 145V for 55 mins. The gels were transferred for 10mins onto a iBlot® Transfer Stack, PVDF regular (Invitrogen) using theInvitrogen iBlot transfer system. PVDF blocking, antibody incubation andwashing were performed using a SNAP i.d. protein detection system(Millipore). The PVDF membranes were then blocked for 30 seconds with0.5% ECL advanced blocking buffer (Fisher). The PVDF's were incubated ina 1/30 dilution of TRPV6 (H-90) primary antibody (Santa Cruz) for 10 minand washed 3 times with 30 mL of TBS-T. The PVDFs were then incubated ina 1/1500 dilution of goat anti-rabbit IgG HRP secondary antibody (SantaCruz) for 10 min and washed 3 times with 30 mL of TBS-T. The TRPV6 bandswere detected with 15 ml of Luminol for 3 mins. Gels were imaged and theband density was quantified with an Alpha Innotech FluroChem imager for10 minutes. All samples were analyzed in triplicate and data werecompared using the Student's t-test and the 95% confidence limit.

Results

As shown in FIG. 26A, the levels of TRPV6 protein were significantlyhigher in blood samples taken from subjects with stage I (p=0.0001) orstage II (p=0.0270) ovarian cancer compared to samples take from healthycontrols. FIG. 26B shows that stage I and stage II ovarian cancerconsidered together (early stage cancers) also exhibit increased levelsof TRPV6 protein (p=0.0006) compared to healthy controls.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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1-14. (canceled)
 15. A method for detecting TRPV6 protein in a samplecomprising: a) contacting the sample with the compound comprising aTransient Receptor Potential Vanilloid 6 (TRPV6)-binding peptideconjugated to a biomolecule, wherein the TRPV6-binding peptide comprisesall or part of a peptide comprising EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ IDNO:1); b) detecting the biomolecule conjugated to the TRPV6-bindingpeptide thereby detecting the TRPV6 protein.
 16. The method of claim 15,wherein the biomolecule comprises a fluorophore and the step ofdetecting the biomolecule comprises detecting the fluorophore.
 17. Themethod of claim 15, wherein the sample is a bodily fluid.
 18. The methodof claim 17, wherein the bodily fluid is blood.
 19. The method of claim15, wherein the biomolecule is detected in vivo.
 20. A method ofidentifying cancer in a sample from a subject comprising: a) detectingTRPV6 mRNA or protein in the sample; b) comparing the amount of TRPV6mRNA or protein in the sample with an amount of TRPV6 mRNA or protein ina control sample, wherein an increased amount of TRPV6 mRNA or proteinin the sample compared to the control is indicative of cancer.
 21. Themethod of claim 20, wherein the step of detecting TRPV6 proteincomprises contacting the sample with the compound comprising a TransientReceptor Potential Vanilloid 6 (TRPV6)-binding peptide conjugated to abiomolecule, wherein the TRPV6-binding peptide comprises all or part ofa peptide comprising (SEQ ID NO: 1) EGKLSSNDTEGGLCKEFLHPSKVDLPR.


22. The method of claim 20, wherein the sample is a bodily fluid. 23.The method of claim 22, wherein the bodily fluid is blood.
 24. Themethod of claim 20, wherein the cancer is stage I cancer or stage IIcancer.
 25. The method of claim 20, wherein the cancer is ovariancancer, breast cancer or prostate cancer.
 26. A method of identifying acancer tumor in a subject comprising: a) administering to the subject acompound comprising a TRPV6-binding peptide or an antibody to TRPV6; b)detecting the TRPV6-binding peptide or antibody to TRPV6 in the subjectthereby detecting TRPV6; c) identifying regions of the subject withincreased levels of TRPV6 relative to a control level, wherein increasedlevels of TRPV6 are indicative of a cancer tumor.
 27. The method ofclaim 26, wherein the compound comprises the compound comprising aTransient Receptor Potential Vanilloid 6 (TRPV6)-binding peptideconjugated to a biomolecule, wherein the TRPV6-binding peptide comprisesall or part of a peptide comprising EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ IDNO:1).
 28. The method of claim 26, wherein the cancer tumor is anovarian cancer tumor, breast cancer tumor or a prostate cancer tumor.29. The method of claim 26, wherein the compound comprising aTRPV6-binding peptide or antibody to TRPV6 comprises a magneticresonance imaging contrast agent and the step of detecting theTRPV6-binding peptide or antibody to TRPV6 comprises magnetic resonanceimaging (MRI).
 30. The method of claim 29, wherein the magneticresonance imaging contrast agent is super paramagnetic iron oxide (SPIO)31. The method of claim 26, wherein the compound comprising aTRPV6-binding peptide or antibody to TRPV6 comprises a fluorophore andthe step of detecting the TRPV6-binding peptide or antibody to TRPV6comprises detecting the fluorophore. 32-37. (canceled)
 38. A compoundcomprising a Transient Receptor Potential Vanilloid 6 (TRPV6)-bindingpeptide conjugated to a biomolecule, wherein the TRPV6-binding peptidecomprises all or part of a peptide comprisingEGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 1).
 39. The compound of claim38, wherein the TRPV6-binding peptide comprises from 9 to 27 amino acidsof SEQ ID NO:
 1. 40. The compound of claim 38, wherein the TRPV6-bindingpeptide comprises a contiguous part of the C-terminal sequence of SEQ IDNO:1.
 41. The compound of claim 40, wherein the TRPV6-binding peptidecomprises at least 9 contiguous amino acids of the C-terminal sequenceof SEQ ID NO:1.
 42. The compound of claim 38, wherein the TRPV6-bindingpeptide has at least 70% identity to the amino acid sequence HPSKVDLPR.43. The compound of claim 38, wherein the TRPV6-binding peptidecomprises the amino acid sequence HPSKVDLPR.
 44. The compound of claim38, wherein the TRPV6-binding peptide has at least 70% identity to theamino acid sequence KEFLHPSKVDLPR.
 45. The compound of claim 38, whereinthe peptide comprises the amino acid sequence KEFLHPSKVDLPR.
 46. Thecompound of claim 38, wherein the TRPV6-binding peptide has at least 70%identity to the amino acid sequence EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ IDNO: 1).
 47. The compound of claim 38, wherein the peptide comprises theamino acid sequence EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 1).
 48. Thecompound of claim 38, wherein the biomolecule comprises a detectablelabel.
 49. The compound of claim 38, wherein the biomolecule comprises atherapeutic agent.
 50. The compound of claim 49, wherein the therapeuticagent is an anti-cancer agent.
 51. A pharmaceutical compositioncomprising the compound of claim 38 and a pharmaceutically acceptablecarrier.
 52. A method of delivering a biomolecule to a cell expressingTRPV6 comprising contacting the cell with the compound of claim
 38. 53.The method of claim 52, wherein the biomolecule comprises a therapeuticagent.
 54. The method of claim 53, wherein the therapeutic agentcomprises an anti-cancer agent.
 55. The method of claim 52, wherein thebiomolecule comprises a detectable label.
 56. The method of claim 55,wherein the detectable label is a fluorophore or a magnetic resonanceimaging contrast agent.
 57. The method of claim 52, wherein the step ofcontacting the cell occurs in vivo.